rowid,run.accession,experiment.accession,sample.accession,study.accession,bioproject,study.title,study.alias,study.type,study.abstract,study.attributes,study.PMIDs,sample.description,sample.title,sample.alias,sample.centername,sample.attributes,GEOsample.title,GEOsample.dataprocessing,GEOsample.source,GEOsample.treatmentprotocol,GEOsample.extractprotocol,GEOsample.growthprotocol,GEOsample.characteristics,GEOsample.accession,experiment.title,experiment.alias,experiment.library_name,experiment.design_description,experiment.library_construction_protocol,experiment.attributes,experiment.library_strategy,experiment.library_source,experiment.library_selection,experiment.library_layout,experiment.platform,experiment.instrument_model,experiment.spot_descriptor,experiment.study_ref,run.title,run.attributes,run.filename,run.semantic_name,run.total_bases,run.total_spots,run.alias,run.read_lengths,run.base_counts,run.r1_length,run.r2_length,run.r3_length,run.r4_length,run.Acount,run.Ccount,run.Gcount,run.Tcount,run.Ncount,run.experiment,run.pool_member,submission.accession,submission.srasource,submission.bioprojectsource,seqdetective.n_mates,seqdetective.mapping_rate.mate1,seqdetective.mapping_rate.mate2,seqdetective.nofeature_rate.mate1,seqdetective.nofeature_rate.mate2,seqdetective.sparsity.mate1,seqdetective.sparsity.mate2,seqdetective.pos_strand_rate.mate1,seqdetective.pos_strand_rate.mate2,seqdetective.readlen.mate1,seqdetective.readlen.mate2,seqdetective.judgement.mate1,seqdetective.judgement.mate2,seqdetective.judgement.reason,platform_family,instrument_generation,read_bias,selection_class,prep_kit,sc_or_bulk,tech_class,technology,tech_variant,submission.bioprojectsource.country,earliest_date,devstage_curation,devstage_curation_coarse,tissue_curation,tissue_curation_coarse 10237,ERR7131169,ERX6698608,ERS8070397,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F8 Nega,SAMEA10418613,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418613|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F8 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 8|organism part:olfactory bulb|sample name:E MTAB 11083:F8 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F8 Nega s,F8 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F22-1_210715_D00404_0538_BCD91CANXX_TCGACGTC-CTAAGCCT_L006_R1_001.fastq.gz,fastq,658093749.0,12903799.0,E MTAB 11083:2874F22 1 210715 D00404 0538 BCD91CANXX TCGACGTC CTAAGCCT L006,0:51 1:0,A:173355262;C:152464129;G:147489024;T:184739885;N:45449,51,0,,,173355262,152464129,147489024,184739885,45449,ERX6698608,ERS8070397,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.78328,,0.15147,,0.71289,,0.53671,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10238,ERR7131170,ERX6698608,ERS8070397,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F8 Nega,SAMEA10418613,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418613|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F8 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 8|organism part:olfactory bulb|sample name:E MTAB 11083:F8 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F8 Nega s,F8 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F22-2_210715_D00404_0538_BCD91CANXX_TCGACGTC-CTAAGCCT_L007_R1_001.fastq.gz,fastq,661103769.0,12962819.0,E MTAB 11083:2874F22 2 210715 D00404 0538 BCD91CANXX TCGACGTC CTAAGCCT L007,0:51 1:0,A:174223249;C:153223284;G:148276805;T:185335092;N:45339,51,0,,,174223249,153223284,148276805,185335092,45339,ERX6698608,ERS8070397,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.78539,,0.15054,,0.70897,,0.5322,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10239,ERR7131167,ERX6698607,ERS8070396,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F8 mCherry GFP,SAMEA10418612,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418612|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F8 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 8|organism part:olfactory bulb|sample name:E MTAB 11083:F8 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F8 mCherry GFP s,F8 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F24-1_210715_D00404_0538_BCD91CANXX_TCGACGTC-TCTCTCCG_L006_R1_001.fastq.gz,fastq,693183636.0,13591836.0,E MTAB 11083:2874F24 1 210715 D00404 0538 BCD91CANXX TCGACGTC TCTCTCCG L006,0:51 1:0,A:183977687;C:159275153;G:153149826;T:196732022;N:48948,51,0,,,183977687,159275153,153149826,196732022,48948,ERX6698607,ERS8070396,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.68942,,0.15593,,0.75828,,0.51858,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10240,ERR7131168,ERX6698607,ERS8070396,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F8 mCherry GFP,SAMEA10418612,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418612|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F8 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 8|organism part:olfactory bulb|sample name:E MTAB 11083:F8 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F8 mCherry GFP s,F8 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F24-2_210715_D00404_0538_BCD91CANXX_TCGACGTC-TCTCTCCG_L007_R1_001.fastq.gz,fastq,696648678.0,13659778.0,E MTAB 11083:2874F24 2 210715 D00404 0538 BCD91CANXX TCGACGTC TCTCTCCG L007,0:51 1:0,A:184971832;C:160126524;G:154012023;T:197490098;N:48201,51,0,,,184971832,160126524,154012023,197490098,48201,ERX6698607,ERS8070396,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.6906,,0.15634,,0.75909,,0.51346,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10241,ERR7131165,ERX6698606,ERS8070395,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F8 mCherry,SAMEA10418611,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418611|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F8 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 8|organism part:olfactory bulb|sample name:E MTAB 11083:F8 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F8 mCherry s,F8 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F23-1_210715_D00404_0538_BCD91CANXX_TCGACGTC-CGTCTAAT_L006_R1_001.fastq.gz,fastq,667760646.0,13093346.0,E MTAB 11083:2874F23 1 210715 D00404 0538 BCD91CANXX TCGACGTC CGTCTAAT L006,0:51 1:0,A:179948739;C:150541918;G:145038845;T:192184092;N:47052,51,0,,,179948739,150541918,145038845,192184092,47052,ERX6698606,ERS8070395,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.71661,,0.19774,,0.74576,,0.52117,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10242,ERR7131166,ERX6698606,ERS8070395,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F8 mCherry,SAMEA10418611,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418611|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F8 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 8|organism part:olfactory bulb|sample name:E MTAB 11083:F8 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F8 mCherry s,F8 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F23-2_210715_D00404_0538_BCD91CANXX_TCGACGTC-CGTCTAAT_L007_R1_001.fastq.gz,fastq,670970484.0,13156284.0,E MTAB 11083:2874F23 2 210715 D00404 0538 BCD91CANXX TCGACGTC CGTCTAAT L007,0:51 1:0,A:180906557;C:151329492;G:145816347;T:192872804;N:45284,51,0,,,180906557,151329492,145816347,192872804,45284,ERX6698606,ERS8070395,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.71682,,0.19902,,0.74517,,0.52618,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10243,ERR7131163,ERX6698605,ERS8070394,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F7 Nega,SAMEA10418610,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418610|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F7 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 7|organism part:olfactory bulb|sample name:E MTAB 11083:F7 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F7 Nega s,F7 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F19-1_210715_D00404_0538_BCD91CANXX_TCGACGTC-GTAAGGAG_L006_R1_001.fastq.gz,fastq,652205238.0,12788338.0,E MTAB 11083:2874F19 1 210715 D00404 0538 BCD91CANXX TCGACGTC GTAAGGAG L006,0:51 1:0,A:173730405;C:149044598;G:145436777;T:183946928;N:46530,51,0,,,173730405,149044598,145436777,183946928,46530,ERX6698605,ERS8070394,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.80595,,0.17309,,0.71003,,0.53696,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10244,ERR7131164,ERX6698605,ERS8070394,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F7 Nega,SAMEA10418610,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418610|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F7 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 7|organism part:olfactory bulb|sample name:E MTAB 11083:F7 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F7 Nega s,F7 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F19-2_210715_D00404_0538_BCD91CANXX_TCGACGTC-GTAAGGAG_L007_R1_001.fastq.gz,fastq,655593423.0,12854773.0,E MTAB 11083:2874F19 2 210715 D00404 0538 BCD91CANXX TCGACGTC GTAAGGAG L007,0:51 1:0,A:174715119;C:149844974;G:146250446;T:184737535;N:45349,51,0,,,174715119,149844974,146250446,184737535,45349,ERX6698605,ERS8070394,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.80585,,0.17497,,0.71078,,0.53767,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10245,ERR7131161,ERX6698604,ERS8070393,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F7 mCherry GFP,SAMEA10418609,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418609|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F7 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 7|organism part:olfactory bulb|sample name:E MTAB 11083:F7 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F7 mCherry GFP s,F7 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F21-1_210715_D00404_0538_BCD91CANXX_TCGACGTC-AAGGAGTA_L006_R1_001.fastq.gz,fastq,661067151.0,12962101.0,E MTAB 11083:2874F21 1 210715 D00404 0538 BCD91CANXX TCGACGTC AAGGAGTA L006,0:51 1:0,A:168281419;C:157701209;G:153487197;T:181550252;N:47074,51,0,,,168281419,157701209,153487197,181550252,47074,ERX6698604,ERS8070393,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.39074,,0.11592,,0.82615,,0.5252,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10246,ERR7131162,ERX6698604,ERS8070393,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F7 mCherry GFP,SAMEA10418609,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418609|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F7 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 7|organism part:olfactory bulb|sample name:E MTAB 11083:F7 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F7 mCherry GFP s,F7 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F21-2_210715_D00404_0538_BCD91CANXX_TCGACGTC-AAGGAGTA_L007_R1_001.fastq.gz,fastq,665709018.0,13053118.0,E MTAB 11083:2874F21 2 210715 D00404 0538 BCD91CANXX TCGACGTC AAGGAGTA L007,0:51 1:0,A:169539998;C:158884603;G:154624803;T:182614264;N:45350,51,0,,,169539998,158884603,154624803,182614264,45350,ERX6698604,ERS8070393,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.38966,,0.1152,,0.8258,,0.53305,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10247,ERR7131159,ERX6698603,ERS8070392,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F7 mCherry,SAMEA10418608,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418608|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F7 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 7|organism part:olfactory bulb|sample name:E MTAB 11083:F7 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F7 mCherry s,F7 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F20-1_210715_D00404_0538_BCD91CANXX_TCGACGTC-ACTGCATA_L006_R1_001.fastq.gz,fastq,657813402.0,12898302.0,E MTAB 11083:2874F20 1 210715 D00404 0538 BCD91CANXX TCGACGTC ACTGCATA L006,0:51 1:0,A:175873489;C:149476129;G:143987770;T:188429588;N:46426,51,0,,,175873489,149476129,143987770,188429588,46426,ERX6698603,ERS8070392,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.70507,,0.2048,,0.75923,,0.52828,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10248,ERR7131160,ERX6698603,ERS8070392,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F7 mCherry,SAMEA10418608,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418608|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F7 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 7|organism part:olfactory bulb|sample name:E MTAB 11083:F7 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F7 mCherry s,F7 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F20-2_210715_D00404_0538_BCD91CANXX_TCGACGTC-ACTGCATA_L007_R1_001.fastq.gz,fastq,660303426.0,12947126.0,E MTAB 11083:2874F20 2 210715 D00404 0538 BCD91CANXX TCGACGTC ACTGCATA L007,0:51 1:0,A:176607119;C:150065781;G:144613110;T:188972809;N:44607,51,0,,,176607119,150065781,144613110,188972809,44607,ERX6698603,ERS8070392,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.70705,,0.20314,,0.75852,,0.52949,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10249,ERR7131157,ERX6698602,ERS8070391,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F6 Nega,SAMEA10418607,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418607|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F6 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 6|organism part:olfactory bulb|sample name:E MTAB 11083:F6 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F6 Nega s,F6 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F16-1_210715_D00404_0538_BCD91CANXX_TGCAGCTA-TCTCTCCG_L006_R1_001.fastq.gz,fastq,669627450.0,13129950.0,E MTAB 11083:2874F16 1 210715 D00404 0538 BCD91CANXX TGCAGCTA TCTCTCCG L006,0:51 1:0,A:177483937;C:154097051;G:148063012;T:189935690;N:47760,51,0,,,177483937,154097051,148063012,189935690,47760,ERX6698602,ERS8070391,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.77183,,0.16001,,0.71467,,0.53352,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10250,ERR7131158,ERX6698602,ERS8070391,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F6 Nega,SAMEA10418607,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418607|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F6 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 6|organism part:olfactory bulb|sample name:E MTAB 11083:F6 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F6 Nega s,F6 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F16-2_210715_D00404_0538_BCD91CANXX_TGCAGCTA-TCTCTCCG_L007_R1_001.fastq.gz,fastq,671615073.0,13168923.0,E MTAB 11083:2874F16 2 210715 D00404 0538 BCD91CANXX TGCAGCTA TCTCTCCG L007,0:51 1:0,A:178109157;C:154619024;G:148595207;T:190246469;N:45216,51,0,,,178109157,154619024,148595207,190246469,45216,ERX6698602,ERS8070391,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.77284,,0.15963,,0.71569,,0.53038,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10251,ERR7131155,ERX6698601,ERS8070390,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F6 mCherry GFP,SAMEA10418606,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418606|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F6 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 6|organism part:olfactory bulb|sample name:E MTAB 11083:F6 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F6 mCherry GFP s,F6 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F18-1_210715_D00404_0538_BCD91CANXX_TCGACGTC-TATCCTCT_L006_R1_001.fastq.gz,fastq,667663542.0,13091442.0,E MTAB 11083:2874F18 1 210715 D00404 0538 BCD91CANXX TCGACGTC TATCCTCT L006,0:51 1:0,A:177646483;C:152747528;G:146388226;T:190833951;N:47354,51,0,,,177646483,152747528,146388226,190833951,47354,ERX6698601,ERS8070390,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.67964,,0.18123,,0.77193,,0.53313,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10252,ERR7131156,ERX6698601,ERS8070390,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F6 mCherry GFP,SAMEA10418606,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418606|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F6 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 6|organism part:olfactory bulb|sample name:E MTAB 11083:F6 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F6 mCherry GFP s,F6 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F18-2_210715_D00404_0538_BCD91CANXX_TCGACGTC-TATCCTCT_L007_R1_001.fastq.gz,fastq,670170141.0,13140591.0,E MTAB 11083:2874F18 2 210715 D00404 0538 BCD91CANXX TCGACGTC TATCCTCT L007,0:51 1:0,A:178425395;C:153363855;G:147033779;T:191300976;N:46136,51,0,,,178425395,153363855,147033779,191300976,46136,ERX6698601,ERS8070390,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.67922,,0.18101,,0.77141,,0.53601,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10253,ERR7131153,ERX6698600,ERS8070389,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F6 mCherry,SAMEA10418605,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418605|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F6 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 6|organism part:olfactory bulb|sample name:E MTAB 11083:F6 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F6 mCherry s,F6 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F17-1_210715_D00404_0538_BCD91CANXX_TCGACGTC-CTCTCTAT_L006_R1_001.fastq.gz,fastq,677617008.0,13286608.0,E MTAB 11083:2874F17 1 210715 D00404 0538 BCD91CANXX TCGACGTC CTCTCTAT L006,0:51 1:0,A:181785899;C:152797687;G:147070676;T:195914808;N:47938,51,0,,,181785899,152797687,147070676,195914808,47938,ERX6698600,ERS8070389,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.68298,,0.1953,,0.76292,,0.5395,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10254,ERR7131154,ERX6698600,ERS8070389,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F6 mCherry,SAMEA10418605,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418605|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F6 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 6|organism part:olfactory bulb|sample name:E MTAB 11083:F6 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F6 mCherry s,F6 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F17-2_210715_D00404_0538_BCD91CANXX_TCGACGTC-CTCTCTAT_L007_R1_001.fastq.gz,fastq,679922973.0,13331823.0,E MTAB 11083:2874F17 2 210715 D00404 0538 BCD91CANXX TCGACGTC CTCTCTAT L007,0:51 1:0,A:182567819;C:153380607;G:147589890;T:196337722;N:46935,51,0,,,182567819,153380607,147589890,196337722,46935,ERX6698600,ERS8070389,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.68391,,0.19726,,0.76299,,0.54099,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10255,ERR7131151,ERX6698599,ERS8070388,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F5 Nega,SAMEA10418604,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418604|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F5 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 5|organism part:olfactory bulb|sample name:E MTAB 11083:F5 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F5 Nega s,F5 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F13-1_210715_D00404_0538_BCD91CANXX_TGCAGCTA-AAGGAGTA_L006_R1_001.fastq.gz,fastq,620316315.0,12163065.0,E MTAB 11083:2874F13 1 210715 D00404 0538 BCD91CANXX TGCAGCTA AAGGAGTA L006,0:51 1:0,A:165967355;C:141072216;G:136931219;T:176301963;N:43562,51,0,,,165967355,141072216,136931219,176301963,43562,ERX6698599,ERS8070388,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.78924,,0.17457,,0.71934,,0.53563,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10256,ERR7131152,ERX6698599,ERS8070388,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F5 Nega,SAMEA10418604,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418604|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F5 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 5|organism part:olfactory bulb|sample name:E MTAB 11083:F5 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F5 Nega s,F5 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F13-2_210715_D00404_0538_BCD91CANXX_TGCAGCTA-AAGGAGTA_L007_R1_001.fastq.gz,fastq,623905338.0,12233438.0,E MTAB 11083:2874F13 2 210715 D00404 0538 BCD91CANXX TGCAGCTA AAGGAGTA L007,0:51 1:0,A:166996253;C:141937637;G:137825206;T:177103504;N:42738,51,0,,,166996253,141937637,137825206,177103504,42738,ERX6698599,ERS8070388,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.78975,,0.17387,,0.72153,,0.54149,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10257,ERR7131149,ERX6698598,ERS8070387,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F5 mCherry GFP,SAMEA10418603,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418603|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F5 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 5|organism part:olfactory bulb|sample name:E MTAB 11083:F5 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F5 mCherry GFP s,F5 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F15-1_210715_D00404_0538_BCD91CANXX_TGCAGCTA-CGTCTAAT_L006_R1_001.fastq.gz,fastq,670154841.0,13140291.0,E MTAB 11083:2874F15 1 210715 D00404 0538 BCD91CANXX TGCAGCTA CGTCTAAT L006,0:51 1:0,A:182457480;C:149175456;G:144548173;T:193927470;N:46262,51,0,,,182457480,149175456,144548173,193927470,46262,ERX6698598,ERS8070387,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.71875,,0.18671,,0.76047,,0.52398,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10258,ERR7131150,ERX6698598,ERS8070387,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F5 mCherry GFP,SAMEA10418603,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418603|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F5 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 5|organism part:olfactory bulb|sample name:E MTAB 11083:F5 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F5 mCherry GFP s,F5 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F15-2_210715_D00404_0538_BCD91CANXX_TGCAGCTA-CGTCTAAT_L007_R1_001.fastq.gz,fastq,674202048.0,13219648.0,E MTAB 11083:2874F15 2 210715 D00404 0538 BCD91CANXX TGCAGCTA CGTCTAAT L007,0:51 1:0,A:183639511;C:150168226;G:145502071;T:194847755;N:44485,51,0,,,183639511,150168226,145502071,194847755,44485,ERX6698598,ERS8070387,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.72111,,0.18813,,0.76378,,0.5258,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10259,ERR7131147,ERX6698597,ERS8070386,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F5 mCherry,SAMEA10418602,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418602|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F5 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 5|organism part:olfactory bulb|sample name:E MTAB 11083:F5 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F5 mCherry s,F5 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F14-1_210715_D00404_0538_BCD91CANXX_TGCAGCTA-CTAAGCCT_L006_R1_001.fastq.gz,fastq,615458259.0,12067809.0,E MTAB 11083:2874F14 1 210715 D00404 0538 BCD91CANXX TGCAGCTA CTAAGCCT L006,0:51 1:0,A:165108582;C:139178883;G:134070672;T:177059757;N:40365,51,0,,,165108582,139178883,134070672,177059757,40365,ERX6698597,ERS8070386,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.69017,,0.1977,,0.77193,,0.53352,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10260,ERR7131148,ERX6698597,ERS8070386,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F5 mCherry,SAMEA10418602,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418602|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F5 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 5|organism part:olfactory bulb|sample name:E MTAB 11083:F5 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F5 mCherry s,F5 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F14-2_210715_D00404_0538_BCD91CANXX_TGCAGCTA-CTAAGCCT_L007_R1_001.fastq.gz,fastq,619061613.0,12138463.0,E MTAB 11083:2874F14 2 210715 D00404 0538 BCD91CANXX TGCAGCTA CTAAGCCT L007,0:51 1:0,A:166167516;C:140058774;G:134972850;T:177822733;N:39740,51,0,,,166167516,140058774,134972850,177822733,39740,ERX6698597,ERS8070386,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.69118,,0.20019,,0.7707,,0.52174,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10261,ERR7131145,ERX6698596,ERS8070385,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F4 Nega,SAMEA10418601,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418601|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F4 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 4|organism part:olfactory bulb|sample name:E MTAB 11083:F4 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F4 Nega s,F4 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F10-1_210715_D00404_0538_BCD91CANXX_TGCAGCTA-TATCCTCT_L006_R1_001.fastq.gz,fastq,652536687.0,12794837.0,E MTAB 11083:2874F10 1 210715 D00404 0538 BCD91CANXX TGCAGCTA TATCCTCT L006,0:51 1:0,A:178033231;C:144661707;G:139876661;T:189921085;N:44003,51,0,,,178033231,144661707,139876661,189921085,44003,ERX6698596,ERS8070385,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.76201,,0.2508,,0.71299,,0.53379,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10262,ERR7131146,ERX6698596,ERS8070385,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F4 Nega,SAMEA10418601,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418601|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F4 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 4|organism part:olfactory bulb|sample name:E MTAB 11083:F4 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F4 Nega s,F4 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F10-2_210715_D00404_0538_BCD91CANXX_TGCAGCTA-TATCCTCT_L007_R1_001.fastq.gz,fastq,655278957.0,12848607.0,E MTAB 11083:2874F10 2 210715 D00404 0538 BCD91CANXX TGCAGCTA TATCCTCT L007,0:51 1:0,A:178938002;C:145332613;G:140527756;T:190437189;N:43397,51,0,,,178938002,145332613,140527756,190437189,43397,ERX6698596,ERS8070385,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.76188,,0.25356,,0.71344,,0.53081,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10263,ERR7131143,ERX6698595,ERS8070384,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F4 mCherry GFP,SAMEA10418600,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418600|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F4 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 4|organism part:olfactory bulb|sample name:E MTAB 11083:F4 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F4 mCherry GFP s,F4 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F12-1_210715_D00404_0538_BCD91CANXX_TGCAGCTA-ACTGCATA_L006_R1_001.fastq.gz,fastq,297921855.0,5841605.0,E MTAB 11083:2874F12 1 210715 D00404 0538 BCD91CANXX TGCAGCTA ACTGCATA L006,0:51 1:0,A:82400724;C:65517496;G:64603058;T:85384480;N:16097,51,0,,,82400724,65517496,64603058,85384480,16097,ERX6698595,ERS8070384,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.72496,,0.20794,,0.81223,,0.52529,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10264,ERR7131144,ERX6698595,ERS8070384,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F4 mCherry GFP,SAMEA10418600,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418600|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F4 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 4|organism part:olfactory bulb|sample name:E MTAB 11083:F4 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F4 mCherry GFP s,F4 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F12-2_210715_D00404_0538_BCD91CANXX_TGCAGCTA-ACTGCATA_L007_R1_001.fastq.gz,fastq,309412971.0,6066921.0,E MTAB 11083:2874F12 2 210715 D00404 0538 BCD91CANXX TGCAGCTA ACTGCATA L007,0:51 1:0,A:85531271;C:68164437;G:67129209;T:88573210;N:14844,51,0,,,85531271,68164437,67129209,88573210,14844,ERX6698595,ERS8070384,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.7245,,0.20724,,0.80468,,0.52791,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10265,ERR7131141,ERX6698594,ERS8070383,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F4 mCherry,SAMEA10418599,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418599|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F4 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 4|organism part:olfactory bulb|sample name:E MTAB 11083:F4 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F4 mCherry s,F4 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F11-1_210715_D00404_0538_BCD91CANXX_TGCAGCTA-GTAAGGAG_L006_R1_001.fastq.gz,fastq,672319791.0,13182741.0,E MTAB 11083:2874F11 1 210715 D00404 0538 BCD91CANXX TGCAGCTA GTAAGGAG L006,0:51 1:0,A:181508034;C:150928549;G:146124085;T:193711358;N:47765,51,0,,,181508034,150928549,146124085,193711358,47765,ERX6698594,ERS8070383,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.71726,,0.19842,,0.74986,,0.53363,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10266,ERR7131142,ERX6698594,ERS8070383,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F4 mCherry,SAMEA10418599,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418599|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F4 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 4|organism part:olfactory bulb|sample name:E MTAB 11083:F4 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F4 mCherry s,F4 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F11-2_210715_D00404_0538_BCD91CANXX_TGCAGCTA-GTAAGGAG_L007_R1_001.fastq.gz,fastq,675486075.0,13244825.0,E MTAB 11083:2874F11 2 210715 D00404 0538 BCD91CANXX TGCAGCTA GTAAGGAG L007,0:51 1:0,A:182462261;C:151688554;G:146902713;T:194385945;N:46602,51,0,,,182462261,151688554,146902713,194385945,46602,ERX6698594,ERS8070383,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.71745,,0.19727,,0.74805,,0.53092,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10267,ERR7131139,ERX6698593,ERS8070382,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F3 Nega,SAMEA10418598,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418598|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F3 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 3|organism part:olfactory bulb|sample name:E MTAB 11083:F3 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F3 Nega s,F3 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F7-1_210715_D00404_0538_BCD91CANXX_CGATCAGT-CGTCTAAT_L006_R1_001.fastq.gz,fastq,630093627.0,12354777.0,E MTAB 11083:2874F7 1 210715 D00404 0538 BCD91CANXX CGATCAGT CGTCTAAT L006,0:51 1:0,A:165132739;C:146179836;G:142796880;T:175940613;N:43559,51,0,,,165132739,146179836,142796880,175940613,43559,ERX6698593,ERS8070382,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.81672,,0.15219,,0.71277,,0.52461,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10268,ERR7131140,ERX6698593,ERS8070382,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F3 Nega,SAMEA10418598,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418598|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F3 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 3|organism part:olfactory bulb|sample name:E MTAB 11083:F3 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F3 Nega s,F3 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F7-2_210715_D00404_0538_BCD91CANXX_CGATCAGT-CGTCTAAT_L007_R1_001.fastq.gz,fastq,632305242.0,12398142.0,E MTAB 11083:2874F7 2 210715 D00404 0538 BCD91CANXX CGATCAGT CGTCTAAT L007,0:51 1:0,A:165832174;C:146751895;G:143384539;T:176293313;N:43321,51,0,,,165832174,146751895,143384539,176293313,43321,ERX6698593,ERS8070382,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.81791,,0.15444,,0.7151,,0.52585,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10269,ERR7131137,ERX6698592,ERS8070381,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F3 mCherry GFP,SAMEA10418597,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418597|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F3 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 3|organism part:olfactory bulb|sample name:E MTAB 11083:F3 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F3 mCherry GFP s,F3 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F9-1_210715_D00404_0538_BCD91CANXX_TGCAGCTA-CTCTCTAT_L006_R1_001.fastq.gz,fastq,660379620.0,12948620.0,E MTAB 11083:2874F9 1 210715 D00404 0538 BCD91CANXX TGCAGCTA CTCTCTAT L006,0:51 1:0,A:178482695;C:147417787;G:143797476;T:190635173;N:46489,51,0,,,178482695,147417787,143797476,190635173,46489,ERX6698592,ERS8070381,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.65635,,0.17778,,0.76899,,0.52957,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10270,ERR7131138,ERX6698592,ERS8070381,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F3 mCherry GFP,SAMEA10418597,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418597|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F3 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 3|organism part:olfactory bulb|sample name:E MTAB 11083:F3 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F3 mCherry GFP s,F3 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F9-2_210715_D00404_0538_BCD91CANXX_TGCAGCTA-CTCTCTAT_L007_R1_001.fastq.gz,fastq,662411307.0,12988457.0,E MTAB 11083:2874F9 2 210715 D00404 0538 BCD91CANXX TGCAGCTA CTCTCTAT L007,0:51 1:0,A:179158323;C:147942300;G:144319925;T:190945370;N:45389,51,0,,,179158323,147942300,144319925,190945370,45389,ERX6698592,ERS8070381,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.65813,,0.17896,,0.77076,,0.52835,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10271,ERR7131135,ERX6698591,ERS8070380,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F3 mCherry,SAMEA10418596,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418596|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F3 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 3|organism part:olfactory bulb|sample name:E MTAB 11083:F3 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F3 mCherry s,F3 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F8-1_210715_D00404_0538_BCD91CANXX_CGATCAGT-TCTCTCCG_L006_R1_001.fastq.gz,fastq,698608353.0,13698203.0,E MTAB 11083:2874F8 1 210715 D00404 0538 BCD91CANXX CGATCAGT TCTCTCCG L006,0:51 1:0,A:188718612;C:156814081;G:151125083;T:201901570;N:49007,51,0,,,188718612,156814081,151125083,201901570,49007,ERX6698591,ERS8070380,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.70185,,0.21547,,0.75588,,0.53853,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10272,ERR7131136,ERX6698591,ERS8070380,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F3 mCherry,SAMEA10418596,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418596|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F3 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 3|organism part:olfactory bulb|sample name:E MTAB 11083:F3 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F3 mCherry s,F3 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F8-2_210715_D00404_0538_BCD91CANXX_CGATCAGT-TCTCTCCG_L007_R1_001.fastq.gz,fastq,699869175.0,13722925.0,E MTAB 11083:2874F8 2 210715 D00404 0538 BCD91CANXX CGATCAGT TCTCTCCG L007,0:51 1:0,A:189192315;C:157176897;G:151498983;T:201952867;N:48113,51,0,,,189192315,157176897,151498983,201952867,48113,ERX6698591,ERS8070380,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.70221,,0.21684,,0.75621,,0.53787,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10273,ERR7131133,ERX6698590,ERS8070379,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F2 Nega,SAMEA10418595,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418595|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F2 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 2|organism part:olfactory bulb|sample name:E MTAB 11083:F2 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F2 Nega s,F2 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F4-1_210715_D00404_0538_BCD91CANXX_CGATCAGT-ACTGCATA_L006_R1_001.fastq.gz,fastq,573812985.0,11251235.0,E MTAB 11083:2874F4 1 210715 D00404 0538 BCD91CANXX CGATCAGT ACTGCATA L006,0:51 1:0,A:150716404;C:132203167;G:129732968;T:161120057;N:40389,51,0,,,150716404,132203167,129732968,161120057,40389,ERX6698590,ERS8070379,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.81899,,0.17302,,0.72281,,0.54315,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10274,ERR7131134,ERX6698590,ERS8070379,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F2 Nega,SAMEA10418595,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418595|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F2 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 2|organism part:olfactory bulb|sample name:E MTAB 11083:F2 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F2 Nega s,F2 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F4-2_210715_D00404_0538_BCD91CANXX_CGATCAGT-ACTGCATA_L007_R1_001.fastq.gz,fastq,576184587.0,11297737.0,E MTAB 11083:2874F4 2 210715 D00404 0538 BCD91CANXX CGATCAGT ACTGCATA L007,0:51 1:0,A:151387408;C:132788210;G:130392043;T:161577529;N:39397,51,0,,,151387408,132788210,130392043,161577529,39397,ERX6698590,ERS8070379,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.81999,,0.17082,,0.72196,,0.54505,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10275,ERR7131131,ERX6698589,ERS8070378,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F2 mCherry GFP,SAMEA10418594,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418594|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F2 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 2|organism part:olfactory bulb|sample name:E MTAB 11083:F2 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F2 mCherry GFP s,F2 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F6-1_210715_D00404_0538_BCD91CANXX_CGATCAGT-CTAAGCCT_L006_R1_001.fastq.gz,fastq,654811032.0,12839432.0,E MTAB 11083:2874F6 1 210715 D00404 0538 BCD91CANXX CGATCAGT CTAAGCCT L006,0:51 1:0,A:177403486;C:146202733;G:142207545;T:188951373;N:45895,51,0,,,177403486,146202733,142207545,188951373,45895,ERX6698589,ERS8070378,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.67854,,0.21407,,0.76104,,0.52713,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10276,ERR7131132,ERX6698589,ERS8070378,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F2 mCherry GFP,SAMEA10418594,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418594|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F2 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 2|organism part:olfactory bulb|sample name:E MTAB 11083:F2 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F2 mCherry GFP s,F2 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F6-2_210715_D00404_0538_BCD91CANXX_CGATCAGT-CTAAGCCT_L007_R1_001.fastq.gz,fastq,656911926.0,12880626.0,E MTAB 11083:2874F6 2 210715 D00404 0538 BCD91CANXX CGATCAGT CTAAGCCT L007,0:51 1:0,A:178119212;C:146730132;G:142725348;T:189293212;N:44022,51,0,,,178119212,146730132,142725348,189293212,44022,ERX6698589,ERS8070378,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.68162,,0.21431,,0.761,,0.5071,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10277,ERR7131129,ERX6698588,ERS8070377,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F2 mCherry,SAMEA10418593,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418593|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F2 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 2|organism part:olfactory bulb|sample name:E MTAB 11083:F2 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F2 mCherry s,F2 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F5-1_210715_D00404_0538_BCD91CANXX_CGATCAGT-AAGGAGTA_L006_R1_001.fastq.gz,fastq,607851558.0,11918658.0,E MTAB 11083:2874F5 1 210715 D00404 0538 BCD91CANXX CGATCAGT AAGGAGTA L006,0:51 1:0,A:164814138;C:136071648;G:132945950;T:173976881;N:42941,51,0,,,164814138,136071648,132945950,173976881,42941,ERX6698588,ERS8070377,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.78967,,0.18905,,0.73241,,0.52347,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10278,ERR7131130,ERX6698588,ERS8070377,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F2 mCherry,SAMEA10418593,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418593|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F2 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 2|organism part:olfactory bulb|sample name:E MTAB 11083:F2 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F2 mCherry s,F2 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F5-2_210715_D00404_0538_BCD91CANXX_CGATCAGT-AAGGAGTA_L007_R1_001.fastq.gz,fastq,610931805.0,11979055.0,E MTAB 11083:2874F5 2 210715 D00404 0538 BCD91CANXX CGATCAGT AAGGAGTA L007,0:51 1:0,A:165714775;C:136822925;G:133684294;T:174667814;N:41997,51,0,,,165714775,136822925,133684294,174667814,41997,ERX6698588,ERS8070377,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.78869,,0.18885,,0.73156,,0.51691,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10279,ERR7131127,ERX6698587,ERS8070376,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F1 Nega,SAMEA10418592,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418592|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F1 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 1|organism part:olfactory bulb|sample name:E MTAB 11083:F1 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F1 Nega s,F1 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F1-1_210715_D00404_0538_BCD91CANXX_CGATCAGT-CTCTCTAT_L006_R1_001.fastq.gz,fastq,619561872.0,12148272.0,E MTAB 11083:2874F1 1 210715 D00404 0538 BCD91CANXX CGATCAGT CTCTCTAT L006,0:51 1:0,A:164385545;C:141746313;G:138163219;T:175223323;N:43472,51,0,,,164385545,141746313,138163219,175223323,43472,ERX6698587,ERS8070376,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.80258,,0.17703,,0.72614,,0.53483,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10280,ERR7131128,ERX6698587,ERS8070376,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F1 Nega,SAMEA10418592,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418592|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F1 Nega|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry /GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 1|organism part:olfactory bulb|sample name:E MTAB 11083:F1 Nega|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F1 Nega s,F1 Nega s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry /GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F1-2_210715_D00404_0538_BCD91CANXX_CGATCAGT-CTCTCTAT_L007_R1_001.fastq.gz,fastq,621481971.0,12185921.0,E MTAB 11083:2874F1 2 210715 D00404 0538 BCD91CANXX CGATCAGT CTCTCTAT L007,0:51 1:0,A:165030774;C:142231469;G:138651078;T:175527080;N:41570,51,0,,,165030774,142231469,138651078,175527080,41570,ERX6698587,ERS8070376,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.80248,,0.17699,,0.726,,0.53711,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10281,ERR7131125,ERX6698586,ERS8070375,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F1 mCherry GFP,SAMEA10418591,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418591|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F1 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 1|organism part:olfactory bulb|sample name:E MTAB 11083:F1 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F1 mCherry GFP s,F1 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F3-1_210715_D00404_0538_BCD91CANXX_CGATCAGT-GTAAGGAG_L006_R1_001.fastq.gz,fastq,651189369.0,12768419.0,E MTAB 11083:2874F3 1 210715 D00404 0538 BCD91CANXX CGATCAGT GTAAGGAG L006,0:51 1:0,A:176398415;C:145659111;G:142137610;T:186948280;N:45953,51,0,,,176398415,145659111,142137610,186948280,45953,ERX6698586,ERS8070375,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.69561,,0.18489,,0.75345,,0.52316,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10282,ERR7131126,ERX6698586,ERS8070375,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F1 mCherry GFP,SAMEA10418591,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418591|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F1 mCherry GFP|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP+|genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 1|organism part:olfactory bulb|sample name:E MTAB 11083:F1 mCherry GFP|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F1 mCherry GFP s,F1 mCherry GFP s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP+,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F3-2_210715_D00404_0538_BCD91CANXX_CGATCAGT-GTAAGGAG_L007_R1_001.fastq.gz,fastq,653398944.0,12811744.0,E MTAB 11083:2874F3 2 210715 D00404 0538 BCD91CANXX CGATCAGT GTAAGGAG L007,0:51 1:0,A:177139734;C:146171766;G:142707146;T:187335475;N:44823,51,0,,,177139734,146171766,142707146,187335475,44823,ERX6698586,ERS8070375,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.69496,,0.18414,,0.75375,,0.52317,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10283,ERR7131123,ERX6698585,ERS8070374,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F1 mCherry,SAMEA10418590,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418590|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F1 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 1|organism part:olfactory bulb|sample name:E MTAB 11083:F1 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F1 mCherry s,F1 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F2-1_210715_D00404_0538_BCD91CANXX_CGATCAGT-TATCCTCT_L006_R1_001.fastq.gz,fastq,613574319.0,12030869.0,E MTAB 11083:2874F2 1 210715 D00404 0538 BCD91CANXX CGATCAGT TATCCTCT L006,0:51 1:0,A:164891629;C:138401759;G:135818073;T:174419828;N:43030,51,0,,,164891629,138401759,135818073,174419828,43030,ERX6698585,ERS8070374,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.79499,,0.21674,,0.73359,,0.52207,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 10284,ERR7131124,ERX6698585,ERS8070374,ERP132560,PRJEB48218,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E-MTAB-11083,Transcriptome Analysis,To investigate the effects of rabies infection on neuronal gene expression we compared gene profiles of rabies infected and non infected GABAergic neurons in the Zebrafish olfactory bulb.,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,,Protocols: EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,F1 mCherry,SAMEA10418590,Friedrich Miescher Institute for Biomedical Research,ENA first public:2021 12 01|ENA last update:2021 12 01|External Id:SAMEA10418590|INSDC center alias:Friedrich Miescher Institute for Biomedical Research|INSDC center name:Friedrich Miescher Institute for Biomedical Research|INSDC first public:2021 12 01T00:23:59Z|INSDC last update:2021 12 01T00:23:59Z|INSDC status:public|Submitter Id:E MTAB 11083:F1 mCherry|age:9|broker name:ArrayExpress|cell type:GABAergic neuron|common name:zebrafish|developmental stage:adult|fraction:mCherry+/GFP |genotype:Tg[gad1b:Gal4 UAS:TVA mCherry]|individual:pool 1|organism part:olfactory bulb|sample name:E MTAB 11083:F1 mCherry|sex:mixed|stimulus:injected with EnvA RV GFP|strain:Ab x Tu x TL x WIK,,,,,,,,,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,E MTAB 11083:F1 mCherry s,F1 mCherry s,Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,EnvA RV GFP was injected to olfactory bulb in Tg[gad1b:Gal4 UAS:TVA mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3 4 days. postwards olfactory bulbs were extracted from 3 5 fish and the samples were pooled. post standard dissociation processes cells were sorted by their fluorescent markers using LSRII. Batch 1 pool 1 to 3 was done 7 days before batch 2 pool 4 to 8. RNA was purified using single cell RNA purification Kit Norgen cat. 51800 mRNA seq libraries were generated using the SmartSeq2 approach Picelli et al Nature protocol 2014 with the following modifications: For cDNA pre amplification up to 10ng of RNA was used as input typically 1 3ng and Reverse Transcription was performed using Superscript IV Thermo Fisher Scientific 50C for 10min 80C for 10min. Amplified cDNA 1ng were converted to indexed sequencing libraries by tagmentation using in house purified Tn5 Picelli et al Genome Research 2014 and Illumina Nextera primers.,Experimental Factor: fraction:mCherry+/GFP ,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2500,,ERP132560,Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb,ENA FIRST PUBLIC:2022 07 07|ENA LAST UPDATE:2022 07 07,2874F2-2_210715_D00404_0538_BCD91CANXX_CGATCAGT-TATCCTCT_L007_R1_001.fastq.gz,fastq,615666798.0,12071898.0,E MTAB 11083:2874F2 2 210715 D00404 0538 BCD91CANXX CGATCAGT TATCCTCT L007,0:51 1:0,A:165536502;C:138952758;G:136375193;T:174760659;N:41686,51,0,,,165536502,138952758,136375193,174760659,41686,ERX6698585,ERS8070374,ERA6757553,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,Friedrich Miescher Institute for Biomedical Research|European Nucleotide Archive,1,0.7941,,0.21645,,0.73494,,0.50761,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,nextera,sc,single_cell_plate,smartseq,,Switzerland,2021-12-01,Adult,Adult,Brain,Nervous System 24927,SRR25557778,SRX21286665,SRS18536778,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant XI,GSM7688794,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant XI,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688794,GSM7688794: Morphant XI; Danio rerio; RNA Seq,GSM7688794 r1,GSM7688794,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-XI_S7_L001_R1_001.fastq.gz,fastq,420092501.0,5668059.0,GSM7688794 r1,0:74.12,A:113006440;C:96725832;G:99320773;T:110915967;N:123489,74,,,,113006440,96725832,99320773,110915967,123489,SRX21286665,SRS18536778,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94757,,0.06229,,0.72364,,0.46676,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24928,SRR25557779,SRX21286665,SRS18536778,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant XI,GSM7688794,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant XI,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688794,GSM7688794: Morphant XI; Danio rerio; RNA Seq,GSM7688794 r1,GSM7688794,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-XI_S7_L002_R1_001.fastq.gz,fastq,421869780.0,5690053.0,GSM7688794 r2,0:74.14,A:113530489;C:97144227;G:99697959;T:111388539;N:108566,74,,,,113530489,97144227,99697959,111388539,108566,SRX21286665,SRS18536778,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94921,,0.06226,,0.72462,,0.4738,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24929,SRR25557780,SRX21286665,SRS18536778,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant XI,GSM7688794,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant XI,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688794,GSM7688794: Morphant XI; Danio rerio; RNA Seq,GSM7688794 r1,GSM7688794,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-XI_S7_L003_R1_001.fastq.gz,fastq,425064659.0,5734041.0,GSM7688794 r3,0:74.13,A:114342253;C:97873100;G:100532599;T:112196433;N:120274,74,,,,114342253,97873100,100532599,112196433,120274,SRX21286665,SRS18536778,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94886,,0.06112,,0.72425,,0.47055,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24930,SRR25557781,SRX21286665,SRS18536778,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant XI,GSM7688794,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant XI,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688794,GSM7688794: Morphant XI; Danio rerio; RNA Seq,GSM7688794 r1,GSM7688794,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-XI_S7_L004_R1_001.fastq.gz,fastq,416990548.0,5624902.0,GSM7688794 r4,0:74.13,A:112153356;C:96009158;G:98613145;T:110097476;N:117413,74,,,,112153356,96009158,98613145,110097476,117413,SRX21286665,SRS18536778,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94869,,0.06229,,0.72506,,0.47222,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24931,SRR25557782,SRX21286664,SRS18536777,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant X,GSM7688793,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant X,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688793,GSM7688793: Morphant X; Danio rerio; RNA Seq,GSM7688793 r1,GSM7688793,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-X_S6_L001_R1_001.fastq.gz,fastq,434485284.0,5881416.0,GSM7688793 r1,0:73.87,A:116640351;C:100176557;G:102659919;T:114799536;N:208921,73,,,,116640351,100176557,102659919,114799536,208921,SRX21286664,SRS18536777,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94533,,0.07176,,0.72464,,0.47632,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24932,SRR25557783,SRX21286664,SRS18536777,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant X,GSM7688793,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant X,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688793,GSM7688793: Morphant X; Danio rerio; RNA Seq,GSM7688793 r1,GSM7688793,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-X_S6_L002_R1_001.fastq.gz,fastq,435203869.0,5886556.0,GSM7688793 r2,0:73.93,A:116869356;C:100363580;G:102830644;T:114973504;N:166785,73,,,,116869356,100363580,102830644,114973504,166785,SRX21286664,SRS18536777,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94542,,0.07203,,0.72421,,0.47733,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24933,SRR25557784,SRX21286664,SRS18536777,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant X,GSM7688793,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant X,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688793,GSM7688793: Morphant X; Danio rerio; RNA Seq,GSM7688793 r1,GSM7688793,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-X_S6_L003_R1_001.fastq.gz,fastq,439897269.0,5951878.0,GSM7688793 r3,0:73.91,A:118101648;C:101426657;G:103990006;T:116184480;N:194478,73,,,,118101648,101426657,103990006,116184480,194478,SRX21286664,SRS18536777,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94486,,0.07224,,0.72448,,0.47915,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24934,SRR25557785,SRX21286664,SRS18536777,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant X,GSM7688793,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant X,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688793,GSM7688793: Morphant X; Danio rerio; RNA Seq,GSM7688793 r1,GSM7688793,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-X_S6_L004_R1_001.fastq.gz,fastq,431385256.0,5836029.0,GSM7688793 r4,0:73.92,A:115804970;C:99459059;G:101962932;T:113975935;N:182360,73,,,,115804970,99459059,101962932,113975935,182360,SRX21286664,SRS18536777,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94432,,0.07229,,0.7261,,0.47463,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24935,SRR25557786,SRX21286663,SRS18536776,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant IX,GSM7688792,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant IX,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688792,GSM7688792: Morphant IX; Danio rerio; RNA Seq,GSM7688792 r1,GSM7688792,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-IX_S16_L001_R1_001.fastq.gz,fastq,513309395.0,6929648.0,GSM7688792 r1,0:74.07,A:137428462;C:118688804;G:122019962;T:134991729;N:180438,74,,,,137428462,118688804,122019962,134991729,180438,SRX21286663,SRS18536776,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94188,,0.07029,,0.73772,,0.47692,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24936,SRR25557787,SRX21286663,SRS18536776,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant IX,GSM7688792,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant IX,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688792,GSM7688792: Morphant IX; Danio rerio; RNA Seq,GSM7688792 r1,GSM7688792,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-IX_S16_L002_R1_001.fastq.gz,fastq,517356735.0,6982196.0,GSM7688792 r2,0:74.10,A:138524037;C:119650852;G:122965491;T:136056171;N:160184,74,,,,138524037,119650852,122965491,136056171,160184,SRX21286663,SRS18536776,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94281,,0.06967,,0.73963,,0.48142,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24937,SRR25557788,SRX21286663,SRS18536776,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant IX,GSM7688792,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant IX,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688792,GSM7688792: Morphant IX; Danio rerio; RNA Seq,GSM7688792 r1,GSM7688792,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-IX_S16_L003_R1_001.fastq.gz,fastq,519422328.0,7010631.0,GSM7688792 r3,0:74.09,A:139040684;C:120128748;G:123529198;T:136551898;N:171800,74,,,,139040684,120128748,123529198,136551898,171800,SRX21286663,SRS18536776,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94297,,0.06942,,0.73726,,0.47499,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24938,SRR25557789,SRX21286663,SRS18536776,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant IX,GSM7688792,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant IX,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688792,GSM7688792: Morphant IX; Danio rerio; RNA Seq,GSM7688792 r1,GSM7688792,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-IX_S16_L004_R1_001.fastq.gz,fastq,511640294.0,6905748.0,GSM7688792 r4,0:74.09,A:136914638;C:118302866;G:121704665;T:134543505;N:174620,74,,,,136914638,118302866,121704665,134543505,174620,SRX21286663,SRS18536776,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94176,,0.07029,,0.73868,,0.47987,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24939,SRR25557790,SRX21286662,SRS18536775,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant VIII,GSM7688791,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant VIII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688791,GSM7688791: Morphant VIII; Danio rerio; RNA Seq,GSM7688791 r1,GSM7688791,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-VIII_S14_L001_R1_001.fastq.gz,fastq,429446821.0,5803178.0,GSM7688791 r1,0:74.00,A:114793535;C:99506379;G:102226914;T:112748761;N:171232,74,,,,114793535,99506379,102226914,112748761,171232,SRX21286662,SRS18536775,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94175,,0.06669,,0.73673,,0.48179,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24940,SRR25557791,SRX21286662,SRS18536775,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant VIII,GSM7688791,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant VIII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688791,GSM7688791: Morphant VIII; Danio rerio; RNA Seq,GSM7688791 r1,GSM7688791,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-VIII_S14_L002_R1_001.fastq.gz,fastq,434629893.0,5870828.0,GSM7688791 r2,0:74.03,A:116216940;C:100703527;G:103463365;T:114092681;N:153380,74,,,,116216940,100703527,103463365,114092681,153380,SRX21286662,SRS18536775,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94143,,0.0676,,0.73791,,0.48091,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24941,SRR25557792,SRX21286662,SRS18536775,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant VIII,GSM7688791,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant VIII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688791,GSM7688791: Morphant VIII; Danio rerio; RNA Seq,GSM7688791 r1,GSM7688791,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-VIII_S14_L003_R1_001.fastq.gz,fastq,435879881.0,5888685.0,GSM7688791 r3,0:74.02,A:116548094;C:100971153;G:103794902;T:114400770;N:164962,74,,,,116548094,100971153,103794902,114400770,164962,SRX21286662,SRS18536775,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94195,,0.06686,,0.73785,,0.48044,,73,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24942,SRR25557793,SRX21286662,SRS18536775,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant VIII,GSM7688791,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant VIII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688791,GSM7688791: Morphant VIII; Danio rerio; RNA Seq,GSM7688791 r1,GSM7688791,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-VIII_S14_L004_R1_001.fastq.gz,fastq,429478475.0,5801978.0,GSM7688791 r4,0:74.02,A:114799280;C:99474677;G:102302775;T:112737475;N:164268,74,,,,114799280,99474677,102302775,112737475,164268,SRX21286662,SRS18536775,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94118,,0.06706,,0.73892,,0.47732,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24943,SRR25557794,SRX21286661,SRS18536774,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant VII,GSM7688790,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant VII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688790,GSM7688790: Morphant VII; Danio rerio; RNA Seq,GSM7688790 r1,GSM7688790,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-VII_S15_L001_R1_001.fastq.gz,fastq,459545188.0,6215897.0,GSM7688790 r1,0:73.93,A:121926568;C:107379723;G:110263606;T:119766429;N:208862,73,,,,121926568,107379723,110263606,119766429,208862,SRX21286661,SRS18536774,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94273,,0.06072,,0.74582,,0.47499,,73,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24944,SRR25557795,SRX21286661,SRS18536774,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant VII,GSM7688790,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant VII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688790,GSM7688790: Morphant VII; Danio rerio; RNA Seq,GSM7688790 r1,GSM7688790,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-VII_S15_L002_R1_001.fastq.gz,fastq,463143624.0,6261406.0,GSM7688790 r2,0:73.97,A:122917997;C:108229793;G:111099867;T:120713978;N:181989,73,,,,122917997,108229793,111099867,120713978,181989,SRX21286661,SRS18536774,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94387,,0.06093,,0.74341,,0.47351,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24945,SRR25557796,SRX21286661,SRS18536774,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant VII,GSM7688790,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant VII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688790,GSM7688790: Morphant VII; Danio rerio; RNA Seq,GSM7688790 r1,GSM7688790,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-VII_S15_L003_R1_001.fastq.gz,fastq,465959664.0,6300929.0,GSM7688790 r3,0:73.95,A:123689364;C:108847593;G:111847868;T:121377463;N:197376,73,,,,123689364,108847593,111847868,121377463,197376,SRX21286661,SRS18536774,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94322,,0.06219,,0.74357,,0.47977,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24946,SRR25557797,SRX21286661,SRS18536774,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Morphant VII,GSM7688790,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing,Morphant VII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant,GSM7688790,GSM7688790: Morphant VII; Danio rerio; RNA Seq,GSM7688790 r1,GSM7688790,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Morphant-VII_S15_L004_R1_001.fastq.gz,fastq,459075430.0,6207363.0,GSM7688790 r4,0:73.96,A:121797426;C:107221116;G:110209684;T:119657308;N:189896,73,,,,121797426,107221116,110209684,119657308,189896,SRX21286661,SRS18536774,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94313,,0.06211,,0.74343,,0.47801,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24947,SRR25557798,SRX21286660,SRS18536773,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control XI,GSM7688787,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control XI,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688787,GSM7688787: Control XI; Danio rerio; RNA Seq,GSM7688787 r1,GSM7688787,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-XI_S2_L001_R1_001.fastq.gz,fastq,439719566.0,5947052.0,GSM7688787 r1,0:73.94,A:117482073;C:102083665;G:104685444;T:115272860;N:195524,73,,,,117482073,102083665,104685444,115272860,195524,SRX21286660,SRS18536773,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94829,,0.06278,,0.72525,,0.46494,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24948,SRR25557799,SRX21286660,SRS18536773,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control XI,GSM7688787,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control XI,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688787,GSM7688787: Control XI; Danio rerio; RNA Seq,GSM7688787 r1,GSM7688787,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-XI_S2_L002_R1_001.fastq.gz,fastq,438533503.0,5927990.0,GSM7688787 r2,0:73.98,A:117199594;C:101810881;G:104402169;T:114946611;N:174248,73,,,,117199594,101810881,104402169,114946611,174248,SRX21286660,SRS18536773,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94887,,0.06387,,0.72827,,0.46616,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24949,SRR25557800,SRX21286660,SRS18536773,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control XI,GSM7688787,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control XI,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688787,GSM7688787: Control XI; Danio rerio; RNA Seq,GSM7688787 r1,GSM7688787,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-XI_S2_L003_R1_001.fastq.gz,fastq,443995554.0,6003540.0,GSM7688787 r3,0:73.96,A:118663936;C:103031716;G:105723574;T:116387834;N:188494,73,,,,118663936,103031716,105723574,116387834,188494,SRX21286660,SRS18536773,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94788,,0.06232,,0.72693,,0.46653,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24950,SRR25557801,SRX21286660,SRS18536773,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control XI,GSM7688787,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control XI,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688787,GSM7688787: Control XI; Danio rerio; RNA Seq,GSM7688787 r1,GSM7688787,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-XI_S2_L004_R1_001.fastq.gz,fastq,435088869.0,5882642.0,GSM7688787 r4,0:73.96,A:116261447;C:100986653;G:103615584;T:114042354;N:182831,73,,,,116261447,100986653,103615584,114042354,182831,SRX21286660,SRS18536773,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94914,,0.06241,,0.72829,,0.4546,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24951,SRR25557802,SRX21286659,SRS18536772,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control X,GSM7688785,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control X,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688785,GSM7688785: Control X; Danio rerio; RNA Seq,GSM7688785 r1,GSM7688785,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-X_S1_L001_R1_001.fastq.gz,fastq,395130702.0,5338263.0,GSM7688785 r1,0:74.02,A:107005793;C:90183031;G:92316770;T:105476481;N:148627,74,,,,107005793,90183031,92316770,105476481,148627,SRX21286659,SRS18536772,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94534,,0.07625,,0.73888,,0.48135,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24952,SRR25557803,SRX21286659,SRS18536772,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control X,GSM7688785,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control X,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688785,GSM7688785: Control X; Danio rerio; RNA Seq,GSM7688785 r1,GSM7688785,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-X_S1_L002_R1_001.fastq.gz,fastq,396764458.0,5358448.0,GSM7688785 r2,0:74.04,A:107513717;C:90540282;G:92658504;T:105917444;N:134511,74,,,,107513717,90540282,92658504,105917444,134511,SRX21286659,SRS18536772,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94374,,0.07709,,0.73878,,0.47615,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24953,SRR25557804,SRX21286659,SRS18536772,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control X,GSM7688785,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control X,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688785,GSM7688785: Control X; Danio rerio; RNA Seq,GSM7688785 r1,GSM7688785,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-X_S1_L003_R1_001.fastq.gz,fastq,399623551.0,5397520.0,GSM7688785 r3,0:74.04,A:108230540;C:91192011;G:93388052;T:106665106;N:147842,74,,,,108230540,91192011,93388052,106665106,147842,SRX21286659,SRS18536772,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94399,,0.07657,,0.73797,,0.4794,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24954,SRR25557805,SRX21286659,SRS18536772,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control X,GSM7688785,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control X,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688785,GSM7688785: Control X; Danio rerio; RNA Seq,GSM7688785 r1,GSM7688785,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-X_S1_L004_R1_001.fastq.gz,fastq,393298978.0,5312101.0,GSM7688785 r4,0:74.04,A:106518845;C:89734564;G:91901232;T:105004490;N:139847,74,,,,106518845,89734564,91901232,105004490,139847,SRX21286659,SRS18536772,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94423,,0.07599,,0.73884,,0.47905,,71,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24955,SRR25557806,SRX21286658,SRS18536771,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control IX,GSM7688783,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control IX,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688783,GSM7688783: Control IX; Danio rerio; RNA Seq,GSM7688783 r1,GSM7688783,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-IX_S22_L001_R1_001.fastq.gz,fastq,366045799.0,4944832.0,GSM7688783 r1,0:74.03,A:98000119;C:84654222;G:86951226;T:96295351;N:144881,74,,,,98000119,84654222,86951226,96295351,144881,SRX21286658,SRS18536771,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94303,,0.07499,,0.73085,,0.47881,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24956,SRR25557807,SRX21286658,SRS18536771,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control IX,GSM7688783,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control IX,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688783,GSM7688783: Control IX; Danio rerio; RNA Seq,GSM7688783 r1,GSM7688783,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-IX_S22_L002_R1_001.fastq.gz,fastq,369429068.0,4988719.0,GSM7688783 r2,0:74.05,A:98907882;C:85446993;G:87729593;T:97216477;N:128123,74,,,,98907882,85446993,87729593,97216477,128123,SRX21286658,SRS18536771,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94261,,0.07335,,0.72969,,0.47867,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24957,SRR25557808,SRX21286658,SRS18536771,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control IX,GSM7688783,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control IX,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688783,GSM7688783: Control IX; Danio rerio; RNA Seq,GSM7688783 r1,GSM7688783,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-IX_S22_L003_R1_001.fastq.gz,fastq,369967330.0,4996710.0,GSM7688783 r3,0:74.04,A:99035743;C:85549878;G:87926587;T:97310812;N:144310,74,,,,99035743,85549878,87926587,97310812,144310,SRX21286658,SRS18536771,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94328,,0.0743,,0.73034,,0.47133,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24958,SRR25557809,SRX21286658,SRS18536771,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control IX,GSM7688783,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control IX,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688783,GSM7688783: Control IX; Danio rerio; RNA Seq,GSM7688783 r1,GSM7688783,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-IX_S22_L004_R1_001.fastq.gz,fastq,365499176.0,4936305.0,GSM7688783 r4,0:74.04,A:97825658;C:84517732;G:86863655;T:96157429;N:134702,74,,,,97825658,84517732,86863655,96157429,134702,SRX21286658,SRS18536771,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94286,,0.07288,,0.72999,,0.4783,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24959,SRR25557810,SRX21286657,SRS18536770,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control VIII,GSM7688782,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control VIII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688782,GSM7688782: Control VIII; Danio rerio; RNA Seq,GSM7688782 r1,GSM7688782,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-VIII_S20_L001_R1_001.fastq.gz,fastq,488585907.0,6585668.0,GSM7688782 r1,0:74.19,A:131727468;C:112349971;G:115189425;T:129203159;N:115884,74,,,,131727468,112349971,115189425,129203159,115884,SRX21286657,SRS18536770,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.93794,,0.07381,,0.7315,,0.47355,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24960,SRR25557811,SRX21286657,SRS18536770,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control VIII,GSM7688782,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control VIII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688782,GSM7688782: Control VIII; Danio rerio; RNA Seq,GSM7688782 r1,GSM7688782,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-VIII_S20_L002_R1_001.fastq.gz,fastq,493766544.0,6653820.0,GSM7688782 r2,0:74.21,A:133135166;C:113551418;G:116398455;T:130575771;N:105734,74,,,,133135166,113551418,116398455,130575771,105734,SRX21286657,SRS18536770,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.93729,,0.07312,,0.7307,,0.47966,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24961,SRR25557812,SRX21286657,SRS18536770,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control VIII,GSM7688782,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control VIII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688782,GSM7688782: Control VIII; Danio rerio; RNA Seq,GSM7688782 r1,GSM7688782,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-VIII_S20_L003_R1_001.fastq.gz,fastq,494935071.0,6670024.0,GSM7688782 r3,0:74.20,A:133406912;C:113769494;G:116771560;T:130871612;N:115493,74,,,,133406912,113769494,116771560,130871612,115493,SRX21286657,SRS18536770,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.93847,,0.07383,,0.73194,,0.47646,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24962,SRR25557813,SRX21286657,SRS18536770,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control VIII,GSM7688782,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control VIII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688782,GSM7688782: Control VIII; Danio rerio; RNA Seq,GSM7688782 r1,GSM7688782,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-VIII_S20_L004_R1_001.fastq.gz,fastq,487644286.0,6572100.0,GSM7688782 r4,0:74.20,A:131423055;C:112092623;G:115067911;T:128945959;N:114738,74,,,,131423055,112092623,115067911,128945959,114738,SRX21286657,SRS18536770,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.93708,,0.0732,,0.73186,,0.4781,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24963,SRR25557814,SRX21286656,SRS18536769,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control VII,GSM7688781,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control VII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688781,GSM7688781: Control VII; Danio rerio; RNA Seq,GSM7688781 r1,GSM7688781,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-VII_S21_L001_R1_001.fastq.gz,fastq,419048369.0,5664168.0,GSM7688781 r1,0:73.98,A:111569757;C:97553687;G:100228004;T:109523480;N:173441,73,,,,111569757,97553687,100228004,109523480,173441,SRX21286656,SRS18536769,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94179,,0.06596,,0.74499,,0.46809,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24964,SRR25557815,SRX21286656,SRS18536769,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control VII,GSM7688781,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control VII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688781,GSM7688781: Control VII; Danio rerio; RNA Seq,GSM7688781 r1,GSM7688781,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-VII_S21_L002_R1_001.fastq.gz,fastq,422419362.0,5707444.0,GSM7688781 r2,0:74.01,A:112476977;C:98366185;G:101046586;T:110369882;N:159732,74,,,,112476977,98366185,101046586,110369882,159732,SRX21286656,SRS18536769,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94275,,0.06539,,0.74523,,0.47247,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24965,SRR25557816,SRX21286656,SRS18536769,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control VII,GSM7688781,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control VII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688781,GSM7688781: Control VII; Danio rerio; RNA Seq,GSM7688781 r1,GSM7688781,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-VII_S21_L003_R1_001.fastq.gz,fastq,424260551.0,5733133.0,GSM7688781 r3,0:74.00,A:112957538;C:98789480;G:101496747;T:110850186;N:166600,74,,,,112957538,98789480,101496747,110850186,166600,SRX21286656,SRS18536769,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94174,,0.06482,,0.74304,,0.46935,,75,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24966,SRR25557817,SRX21286656,SRS18536769,SRP453884,PRJNA1003026,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq],GSE240238,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Ten samples were analysed in total all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.,parent bioproject:PRJNA1003022,pubmed:38017520,,Control VII,GSM7688781,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control|geo loc name:missing|collection date:missing,Control VII,We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence “CTGTCTCTTATACACATCT” from the 3’ end using default parameters before trimming bases from the 5’ end selecting an end minimum quality value Phred score of 32 and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting clustering by features using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.,Spinal Cord,The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5’ TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5’ ACGTATCCTGTGTTGTTTCGGGCAT plus 5 ng/nl of a control zebrafish p53 morpholino 5’ GCGCCATTGCTTTGCAAGAATTG into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools. Morpholino injections always produce a spectrum of phenotypes since it is hard to ensure that every cell receives the same dose. Therefore prior to processing for FACS at 27 hpf we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer’s instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals so this could not be used to stage injected embryos. Instead these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle head size and eye size as prim staged uninjected control embryos.,tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:Wild type|treatment:Uninjected control,GSM7688781,GSM7688781: Control VII; Danio rerio; RNA Seq,GSM7688781 r1,GSM7688781,1,Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 30 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT Qiagen RNeasy Micro Kit 74004 plus 143 mMβ mercaptoethanol. Sorted cells were stored at 80oC prior to RNA extraction. Frozen FAC sorted cell lysates were removed from storage at 80oC and thawed in a 37oC waterbath before transferring to sterile microcentrifuge tubes. If necessary sample volumes were completed to 250 µl with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific 10977035. 750 µl TRIzol LS Reagent ThermoFisher Scientific 10296028 was added to each 250 µl sample before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific A33248 before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit Qiagen 74004 before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent 5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara 634888 and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent 5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit v2.5 75 cycles 20024906.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP453884,,loader:fastq load.py,Control-VII_S21_L004_R1_001.fastq.gz,fastq,417875320.0,5646817.0,GSM7688781 r4,0:74.00,A:111226895;C:97273168;G:100030641;T:109182520;N:162096,74,,,,111226895,97273168,100030641,109182520,162096,SRX21286656,SRS18536769,SRA1688461,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.94352,,0.06524,,0.74442,,0.47095,,74,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,nextera,sc,single_cell_plate,smartseq,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 26558,SRR26173859,SRX21885960,SRS18977085,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F17 R1,GSM7804200,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F17 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804200,GSM7804200: V2a sample2 354 F17 R1; Danio rerio; RNA Seq,GSM7804200 r1,GSM7804200,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F17_R1.fastq.gz,fastq,26979490.0,627430.0,GSM7804200 r1,0:43,A:7493119;C:5872644;G:6013311;T:7600416;N:0,43,,,,7493119,5872644,6013311,7600416,0,SRX21885960,SRS18977085,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.82979,,0.33034,,0.94253,,0.53995,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26559,SRR26173860,SRX21885959,SRS18977083,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F16 R1,GSM7804199,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F16 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804199,GSM7804199: V2a sample2 354 F16 R1; Danio rerio; RNA Seq,GSM7804199 r1,GSM7804199,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F16_R1.fastq.gz,fastq,26312130.0,611910.0,GSM7804199 r1,0:43,A:7117249;C:5957278;G:6103577;T:7134026;N:0,43,,,,7117249,5957278,6103577,7134026,0,SRX21885959,SRS18977083,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87071,,0.23579,,0.90678,,0.52226,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26560,SRR26173861,SRX21885958,SRS18977084,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F15 R1,GSM7804198,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F15 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804198,GSM7804198: V2a sample2 354 F15 R1; Danio rerio; RNA Seq,GSM7804198 r1,GSM7804198,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F15_R1.fastq.gz,fastq,32073872.0,745904.0,GSM7804198 r1,0:43,A:8695962;C:7255833;G:7422516;T:8699561;N:0,43,,,,8695962,7255833,7422516,8699561,0,SRX21885958,SRS18977084,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.86262,,0.24391,,0.90881,,0.52511,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26561,SRR26173862,SRX21885957,SRS18977081,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F14 R1,GSM7804197,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F14 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804197,GSM7804197: V2a sample2 354 F14 R1; Danio rerio; RNA Seq,GSM7804197 r1,GSM7804197,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F14_R1.fastq.gz,fastq,31158875.0,724625.0,GSM7804197 r1,0:43,A:8769852;C:6707813;G:6866255;T:8814955;N:0,43,,,,8769852,6707813,6866255,8814955,0,SRX21885957,SRS18977081,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8739,,0.27254,,0.9246,,0.54185,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26562,SRR26173863,SRX21885956,SRS18977082,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F13 R1,GSM7804196,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F13 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804196,GSM7804196: V2a sample2 354 F13 R1; Danio rerio; RNA Seq,GSM7804196 r1,GSM7804196,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F13_R1.fastq.gz,fastq,41289804.0,960228.0,GSM7804196 r1,0:43,A:11114918;C:9358398;G:9568761;T:11247727;N:0,43,,,,11114918,9358398,9568761,11247727,0,SRX21885956,SRS18977082,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.82861,,0.29409,,0.93235,,0.49485,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26563,SRR26173864,SRX21885955,SRS18977079,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F12 R1,GSM7804195,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F12 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804195,GSM7804195: V2a sample2 354 F12 R1; Danio rerio; RNA Seq,GSM7804195 r1,GSM7804195,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F12_R1.fastq.gz,fastq,30569259.0,710913.0,GSM7804195 r1,0:43,A:8286806;C:6854804;G:7028453;T:8399196;N:0,43,,,,8286806,6854804,7028453,8399196,0,SRX21885955,SRS18977079,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.80426,,0.29136,,0.94123,,0.54305,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26564,SRR26173865,SRX21885954,SRS18977080,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F11 R1,GSM7804194,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F11 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804194,GSM7804194: V2a sample2 354 F11 R1; Danio rerio; RNA Seq,GSM7804194 r1,GSM7804194,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F11_R1.fastq.gz,fastq,29626097.0,688979.0,GSM7804194 r1,0:43,A:8063765;C:6645347;G:6803778;T:8113207;N:0,43,,,,8063765,6645347,6803778,8113207,0,SRX21885954,SRS18977080,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.84972,,0.26423,,0.9234,,0.54087,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26565,SRR26173866,SRX21885953,SRS18977078,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F10 R1,GSM7804193,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F10 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804193,GSM7804193: V2a sample2 354 F10 R1; Danio rerio; RNA Seq,GSM7804193 r1,GSM7804193,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F10_R1.fastq.gz,fastq,24594409.0,571963.0,GSM7804193 r1,0:43,A:6605642;C:5535165;G:5682586;T:6771016;N:0,43,,,,6605642,5535165,5682586,6771016,0,SRX21885953,SRS18977078,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.77683,,0.28872,,0.94633,,0.52513,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26566,SRR26173867,SRX21885952,SRS18977077,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E9 R1,GSM7804168,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E9 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804168,GSM7804168: V2a sample2 354 E9 R1; Danio rerio; RNA Seq,GSM7804168 r1,GSM7804168,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E9_R1.fastq.gz,fastq,33259941.0,773487.0,GSM7804168 r1,0:43,A:9004615;C:7506729;G:7674909;T:9073688;N:0,43,,,,9004615,7506729,7674909,9073688,0,SRX21885952,SRS18977077,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.85528,,0.32094,,0.89217,,0.50224,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26567,SRR26173868,SRX21885951,SRS18977076,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E8 R1,GSM7804167,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E8 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804167,GSM7804167: V2a sample2 354 E8 R1; Danio rerio; RNA Seq,GSM7804167 r1,GSM7804167,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E8_R1.fastq.gz,fastq,45348316.0,1054612.0,GSM7804167 r1,0:43,A:12143243;C:10459395;G:10612458;T:12133220;N:0,43,,,,12143243,10459395,10612458,12133220,0,SRX21885951,SRS18977076,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8783,,0.19318,,0.88572,,0.49101,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26568,SRR26173869,SRX21885950,SRS18977075,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E7 R1,GSM7804166,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E7 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804166,GSM7804166: V2a sample2 354 E7 R1; Danio rerio; RNA Seq,GSM7804166 r1,GSM7804166,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E7_R1.fastq.gz,fastq,30522002.0,709814.0,GSM7804166 r1,0:43,A:8167984;C:7033955;G:7127866;T:8192197;N:0,43,,,,8167984,7033955,7127866,8192197,0,SRX21885950,SRS18977075,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87804,,0.20762,,0.88418,,0.51381,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System 26569,SRR26173870,SRX21885949,SRS18977074,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow intermediate or fast subtypes. Furthermore we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function connectivity and behavior our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E6 R1,GSM7804165,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E6 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio Ensembl GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5 R core team 2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804165,GSM7804165: V2a sample2 354 E6 R1; Danio rerio; RNA Seq,GSM7804165 r1,GSM7804165,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl 2.9 KCl 2.1 CaCl2 1.2 MgCl2 10 HEPES and 10 glucose with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line and two samples were prepared from the Tgchx10:GFP line. For each sample 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher #11039021 osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml 280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher #65 0880 92. Using fluorescence activated cell sorting FACs cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100 2 U/ml RNase inhibitor and immediately snap frozen on ice then stored at 80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E6_R1.fastq.gz,fastq,39095858.0,909206.0,GSM7804165 r1,0:43,A:10597068;C:8804712;G:8974908;T:10719170;N:0,43,,,,10597068,8804712,8974908,10719170,0,SRX21885949,SRS18977074,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8093,,0.38297,,0.91545,,0.56195,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System