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RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E MTAB 9113:sibling 3 p", "sibling 3 p", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "Experimental Factor: genotype:wild type genotype", "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP121885", "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19", "UCLGNS1141-gfp-sibs-3_S6_R1.fastq.gz UCLGNS1141-gfp-sibs-3_S6_R2.fastq.gz", "fastq fastq", null, null, "E MTAB 9113:UCLGNS1141 gfp sibs 3 S6 R", "0:81 1:81", "A:1144578958;C:1113109875;G:1138809559;T:1117038434;N:213932", 81, 81, null, null, 1144578958, 1113109875, 1138809559, 1117038434, 213932, "ERX4136409", "ERS4580819", "ERA2625401", "Centre for Developmental Neurobiology King", "Centre for Developmental Neurobiology King", 2, 0.96684, 0.96492, 0.03244, 0.0317, 0.71236, 0.71514, 0.45871, 0.46189, 81, 81, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2020-05-22", "Multi-stage", "Multi-stage", "Embryo Imprecise", "All anatomical structures"], [9893, "ERR4172794", "ERX4136408", "ERS4580818", "ERP121885", "PRJEB38455", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E-MTAB-9113", "Transcriptome Analysis", "RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22", null, "Protocols: Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "sibling 2", "SAMEA6853228", "Centre for Developmental Neurobiology King's College London", "ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853228|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sibling 2|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|sample name:E MTAB 9113:sibling 2|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E MTAB 9113:sibling 2 p", "sibling 2 p", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "Experimental Factor: genotype:wild type genotype", "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP121885", "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19", "UCLGNS1141-gfp-sibs-2_S4_R1.fastq.gz UCLGNS1141-gfp-sibs-2_S4_R2.fastq.gz", "fastq fastq", null, null, "E MTAB 9113:UCLGNS1141 gfp sibs 2 S4 R", "0:81 1:81", "A:1056280368;C:1036355469;G:1045429486;T:1035773622;N:189389", 81, 81, null, null, 1056280368, 1036355469, 1045429486, 1035773622, 189389, "ERX4136408", "ERS4580818", "ERA2625401", "Centre for Developmental Neurobiology King", "Centre for Developmental Neurobiology King", 2, 0.95511, 0.95696, 0.02941, 0.02903, 0.71492, 0.71628, 0.45765, 0.46537, 81, 81, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2020-05-22", "Multi-stage", "Multi-stage", "Embryo Imprecise", "All anatomical structures"], [9894, "ERR4172793", "ERX4136407", "ERS4580817", "ERP121885", "PRJEB38455", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E-MTAB-9113", "Transcriptome Analysis", "RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22", null, "Protocols: Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "sibling 1", "SAMEA6853227", "Centre for Developmental Neurobiology King's College London", "ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853227|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sibling 1|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|sample name:E MTAB 9113:sibling 1|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E MTAB 9113:sibling 1 p", "sibling 1 p", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "Experimental Factor: genotype:wild type genotype", "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP121885", "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19", "UCLGNS1141-gfp-sibs-1_S2_R1.fastq.gz UCLGNS1141-gfp-sibs-1_S2_R2.fastq.gz", "fastq fastq", null, null, "E MTAB 9113:UCLGNS1141 gfp sibs 1 S2 R", "0:81 1:81", "A:1121176859;C:1093927654;G:1108608188;T:1101082277;N:209264", 81, 81, null, null, 1121176859, 1093927654, 1108608188, 1101082277, 209264, "ERX4136407", "ERS4580817", "ERA2625401", "Centre for Developmental Neurobiology King", "Centre for Developmental Neurobiology King", 2, 0.95795, 0.96004, 0.02885, 0.02854, 0.71648, 0.71756, 0.44348, 0.43956, 81, 81, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2020-05-22", "Multi-stage", "Multi-stage", "Embryo Imprecise", "All anatomical structures"], [9895, "ERR4172792", "ERX4136406", "ERS4580816", "ERP121885", "PRJEB38455", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E-MTAB-9113", "Transcriptome Analysis", "RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22", null, "Protocols: Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "sfpq 3", "SAMEA6853226", "Centre for Developmental Neurobiology King's College London", "ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853226|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sfpq 3|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:sfpq / |sample name:E MTAB 9113:sfpq 3|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E MTAB 9113:sfpq 3 p", "sfpq 3 p", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "Experimental Factor: genotype:sfpq / ", "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP121885", "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19", "UCLGNS1141-gfp-neg_3_S5_R1.fastq.gz UCLGNS1141-gfp-neg_3_S5_R2.fastq.gz", "fastq fastq", null, null, "E MTAB 9113:UCLGNS1141 gfp neg 3 S5 R", "0:81 1:81", "A:845849249;C:772302798;G:911660213;T:787409272;N:154042", 81, 81, null, null, 845849249, 772302798, 911660213, 787409272, 154042, "ERX4136406", "ERS4580816", "ERA2625401", "Centre for Developmental Neurobiology King", "Centre for Developmental Neurobiology King", 2, 0.96298, 0.95486, 0.03415, 0.03476, 0.7167, 0.73503, 0.4665, 0.45947, 81, 81, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2020-05-22", "Multi-stage", "Multi-stage", "Embryo Imprecise", "All anatomical structures"], [9896, "ERR4172791", "ERX4136405", "ERS4580815", "ERP121885", "PRJEB38455", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E-MTAB-9113", "Transcriptome Analysis", "RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22", null, "Protocols: Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "sfpq 2", "SAMEA6853225", "Centre for Developmental Neurobiology King's College London", "ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853225|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sfpq 2|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:sfpq / |sample name:E MTAB 9113:sfpq 2|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E MTAB 9113:sfpq 2 p", "sfpq 2 p", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "Experimental Factor: genotype:sfpq / ", "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP121885", "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19", "UCLGNS1141-gfp-neg-2_S3_R1.fastq.gz UCLGNS1141-gfp-neg-2_S3_R2.fastq.gz", "fastq fastq", null, null, "E MTAB 9113:UCLGNS1141 gfp neg 2 S3 R", "0:81 1:81", "A:961156886;C:917011292;G:942713581;T:936109877;N:157724", 81, 81, null, null, 961156886, 917011292, 942713581, 936109877, 157724, "ERX4136405", "ERS4580815", "ERA2625401", "Centre for Developmental Neurobiology King", "Centre for Developmental Neurobiology King", 2, 0.96276, 0.96147, 0.03436, 0.03382, 0.71892, 0.72021, 0.4581, 0.46402, 81, 81, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2020-05-22", "Multi-stage", "Multi-stage", "Embryo Imprecise", "All anatomical structures"], [9897, "ERR4172790", "ERX4136404", "ERS4580814", "ERP121885", "PRJEB38455", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E-MTAB-9113", "Transcriptome Analysis", "RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22", null, "Protocols: Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "sfpq 1", "SAMEA6853224", "Centre for Developmental Neurobiology King's College London", "ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853224|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sfpq 1|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:sfpq / |sample name:E MTAB 9113:sfpq 1|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "E MTAB 9113:sfpq 1 p", "sfpq 1 p", "RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "Embryos were collected from in crosses of sfpq+/  adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion", "Experimental Factor: genotype:sfpq / ", "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP121885", "Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq /  zebrafish embryos and siblings at 24 hpf", "ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19", "UCLGNS1141-gfp-neg-1_S1_R1.fastq.gz UCLGNS1141-gfp-neg-1_S1_R2.fastq.gz", "fastq fastq", null, null, "E MTAB 9113:UCLGNS1141 gfp neg 1 S1 R", "0:81 1:81", "A:1046317524;C:1014946275;G:1036910299;T:1023619857;N:185237", 81, 81, null, null, 1046317524, 1014946275, 1036910299, 1023619857, 185237, "ERX4136404", "ERS4580814", "ERA2625401", "Centre for Developmental Neurobiology King", "Centre for Developmental Neurobiology King", 2, 0.96015, 0.96173, 0.03114, 0.03098, 0.7175, 0.71865, 0.46666, 0.46399, 81, 81, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2020-05-22", "Multi-stage", "Multi-stage", "Embryo Imprecise", "All anatomical structures"], [11130, "ERR9979395", "ERX9520370", "ERS12499848", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish.  Here  we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief  2 hpf embryos were transferred to petri dishes with sterile E3 medium  supplemented with ampicillin 100 \u03bcg/mL  kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf  the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min  followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium  transferred to flasks and incubated at 28\u00b0C. The viability was monitored  and sterile medium was refreshed daily. At day 4  the hatched embryos were used for the sterility validation. A day 5  germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate  four replicates  i.e. eighty larvae in total  were used for each condition. post the exposure  zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly  zebrafish larvae were euthanized with 0.01% of tricaine for 5 min  collected in 1.5 mL tube  and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL  followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT  followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh  with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III  BD Biosciences  NJ  USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN  Kanther M  Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3  1862 1875 2008. 2. Bresciani E  Broadbridge E  Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae  MethodsX. 5  1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab  Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics  Pleasanton  USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly  GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads  a Master Mix containing cells  and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation  the Gel Bead was dissolved  primers containing an Illumina TruSeq Read 1read 1 sequencing primer  16 nt 10x Barcode  12 nt unique molecular identifier UMI and 30 nt polydT sequence were released  and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics  16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation  primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded  full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture  which included leftover biochemical reagents and primers. Barcoded  full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5  P7  i7 and i5 sample indexes  and TruSeq Read 2 read 2 primer sequence were added via End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 2 WT GO+BA", "SAMEA110401656", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401656|INSDC center alias:Institute of Environmental Medicine  Karolinska Institutet|INSDC center name:Institute of Environmental Medicine  Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 2 WT GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 2 WT GO+BA|sex:not available|stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 2 WT GO+BA p", "Sample 2 WT GO+BA p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief  2 hpf embryos were transferred to petri dishes with sterile E3 medium  supplemented with ampicillin 100 \u03bcg/mL  kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf  the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min  followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium  transferred to flasks and incubated at 28\u00b0C. The viability was monitored  and sterile medium was refreshed daily. At day 4  the hatched embryos were used for the sterility validation. A day 5  germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate  four replicates  i.e. eighty larvae in total  were used for each condition. post the exposure  zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly  zebrafish larvae were euthanized with 0.01% of tricaine for 5 min  collected in 1.5 mL tube  and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL  followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT  followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh  with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III  BD Biosciences  NJ  USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN  Kanther M  Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3  1862 1875 2008. 2. Bresciani E  Broadbridge E  Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae  MethodsX. 5  1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab  Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics  Pleasanton  USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol.  Briefly  GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads  a Master Mix containing cells  and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation  the Gel Bead was dissolved  primers containing an Illumina TruSeq Read 1read 1 sequencing primer  16 nt 10x Barcode  12 nt unique molecular identifier UMI and 30 nt polydT sequence were released  and the co partitioned cells were lysed.  The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics  16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation  primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded  full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture  which included leftover biochemical reagents and primers. Barcoded  full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5  P7  i7 and i5 sample indexes  and TruSeq Read 2 read 2 primer sequence were added via End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7002_S2_L002_R1_001.fastq.gz P22202_7002_S2_L002_R2_001.fastq.gz", "fastq fastq", 18144406998.0, 153766161.0, "E MTAB 11984:P22202 7002 S2 L002", "0:28 1:90", "A:5277424121;C:3880928155;G:4118706097;T:4866649878;N:698747", 28, 90, null, null, 5277424121, 3880928155, 4118706097, 4866649878, 698747, "ERX9520370", "ERS12499848", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.0109, 0.90145, 0.0053, 0.21373, 0.98746, 0.79423, 0.41198, 0.54447, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11131, "ERR9979394", "ERX9520370", "ERS12499848", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish.  Here  we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief  2 hpf embryos were transferred to petri dishes with sterile E3 medium  supplemented with ampicillin 100 \u03bcg/mL  kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf  the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min  followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium  transferred to flasks and incubated at 28\u00b0C. The viability was monitored  and sterile medium was refreshed daily. At day 4  the hatched embryos were used for the sterility validation. A day 5  germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate  four replicates  i.e. eighty larvae in total  were used for each condition. post the exposure  zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly  zebrafish larvae were euthanized with 0.01% of tricaine for 5 min  collected in 1.5 mL tube  and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL  followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT  followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh  with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III  BD Biosciences  NJ  USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN  Kanther M  Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3  1862 1875 2008. 2. Bresciani E  Broadbridge E  Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae  MethodsX. 5  1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab  Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics  Pleasanton  USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly  GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads  a Master Mix containing cells  and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation  the Gel Bead was dissolved  primers containing an Illumina TruSeq Read 1read 1 sequencing primer  16 nt 10x Barcode  12 nt unique molecular identifier UMI and 30 nt polydT sequence were released  and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics  16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation  primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded  full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture  which included leftover biochemical reagents and primers. Barcoded  full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5  P7  i7 and i5 sample indexes  and TruSeq Read 2 read 2 primer sequence were added via End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 2 WT GO+BA", "SAMEA110401656", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401656|INSDC center alias:Institute of Environmental Medicine  Karolinska Institutet|INSDC center name:Institute of Environmental Medicine  Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 2 WT GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 2 WT GO+BA|sex:not available|stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 2 WT GO+BA p", "Sample 2 WT GO+BA p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief  2 hpf embryos were transferred to petri dishes with sterile E3 medium  supplemented with ampicillin 100 \u03bcg/mL  kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf  the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min  followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium  transferred to flasks and incubated at 28\u00b0C. The viability was monitored  and sterile medium was refreshed daily. At day 4  the hatched embryos were used for the sterility validation. A day 5  germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate  four replicates  i.e. eighty larvae in total  were used for each condition. post the exposure  zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly  zebrafish larvae were euthanized with 0.01% of tricaine for 5 min  collected in 1.5 mL tube  and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL  followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT  followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh  with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III  BD Biosciences  NJ  USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN  Kanther M  Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3  1862 1875 2008. 2. Bresciani E  Broadbridge E  Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae  MethodsX. 5  1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab  Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics  Pleasanton  USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol.  Briefly  GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads  a Master Mix containing cells  and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation  the Gel Bead was dissolved  primers containing an Illumina TruSeq Read 1read 1 sequencing primer  16 nt 10x Barcode  12 nt unique molecular identifier UMI and 30 nt polydT sequence were released  and the co partitioned cells were lysed.  The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics  16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation  primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded  full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture  which included leftover biochemical reagents and primers. Barcoded  full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5  P7  i7 and i5 sample indexes  and TruSeq Read 2 read 2 primer sequence were added via End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7002_S2_L001_R1_001.fastq.gz P22202_7002_S2_L001_R2_001.fastq.gz", "fastq fastq", 18458951574.0, 156431793.0, "E MTAB 11984:P22202 7002 S2 L001", "0:28 1:90", "A:5372920926;C:3946687814;G:4185647697;T:4953205454;N:489683", 28, 90, null, null, 5372920926, 3946687814, 4185647697, 4953205454, 489683, "ERX9520370", "ERS12499848", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01037, 0.90168, 0.0048, 0.21246, 0.98752, 0.79297, 0.42601, 0.54996, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11132, "ERR9979392", "ERX9520369", "ERS12499847", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish.  Here  we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief  2 hpf embryos were transferred to petri dishes with sterile E3 medium  supplemented with ampicillin 100 \u03bcg/mL  kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf  the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min  followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium  transferred to flasks and incubated at 28\u00b0C. The viability was monitored  and sterile medium was refreshed daily. At day 4  the hatched embryos were used for the sterility validation. A day 5  germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate  four replicates  i.e. eighty larvae in total  were used for each condition. post the exposure  zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly  zebrafish larvae were euthanized with 0.01% of tricaine for 5 min  collected in 1.5 mL tube  and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL  followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT  followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh  with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III  BD Biosciences  NJ  USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN  Kanther M  Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3  1862 1875 2008. 2. Bresciani E  Broadbridge E  Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae  MethodsX. 5  1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab  Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics  Pleasanton  USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly  GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads  a Master Mix containing cells  and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation  the Gel Bead was dissolved  primers containing an Illumina TruSeq Read 1read 1 sequencing primer  16 nt 10x Barcode  12 nt unique molecular identifier UMI and 30 nt polydT sequence were released  and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics  16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation  primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded  full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture  which included leftover biochemical reagents and primers. Barcoded  full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5  P7  i7 and i5 sample indexes  and TruSeq Read 2 read 2 primer sequence were added via End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 1 WT control", "SAMEA110401655", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401655|INSDC center alias:Institute of Environmental Medicine  Karolinska Institutet|INSDC center name:Institute of Environmental Medicine  Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 1 WT control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 1 WT control|sex:not available|stimulus:n1|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 1 WT control p", "Sample 1 WT control p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief  2 hpf embryos were transferred to petri dishes with sterile E3 medium  supplemented with ampicillin 100 \u03bcg/mL  kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf  the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min  followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium  transferred to flasks and incubated at 28\u00b0C. The viability was monitored  and sterile medium was refreshed daily. At day 4  the hatched embryos were used for the sterility validation. A day 5  germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate  four replicates  i.e. eighty larvae in total  were used for each condition. post the exposure  zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly  zebrafish larvae were euthanized with 0.01% of tricaine for 5 min  collected in 1.5 mL tube  and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL  followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT  followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh  with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III  BD Biosciences  NJ  USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN  Kanther M  Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3  1862 1875 2008. 2. Bresciani E  Broadbridge E  Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae  MethodsX. 5  1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab  Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics  Pleasanton  USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol.  Briefly  GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads  a Master Mix containing cells  and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation  the Gel Bead was dissolved  primers containing an Illumina TruSeq Read 1read 1 sequencing primer  16 nt 10x Barcode  12 nt unique molecular identifier UMI and 30 nt polydT sequence were released  and the co partitioned cells were lysed.  The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics  16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation  primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded  full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture  which included leftover biochemical reagents and primers. Barcoded  full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5  P7  i7 and i5 sample indexes  and TruSeq Read 2 read 2 primer sequence were added via End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:n1", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7001_S1_L001_R1_001.fastq.gz P22202_7001_S1_L001_R2_001.fastq.gz", "fastq fastq", 19396994226.0, 164381307.0, "E MTAB 11984:P22202 7001 S1 L001", "0:28 1:90", "A:5665199484;C:4091196594;G:4415211076;T:5224866754;N:520318", 28, 90, null, null, 5665199484, 4091196594, 4415211076, 5224866754, 520318, "ERX9520369", "ERS12499847", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01093, 0.89238, 0.00557, 0.232, 0.98764, 0.79444, 0.40806, 0.54781, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11133, "ERR9979393", "ERX9520369", "ERS12499847", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish.  Here  we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief  2 hpf embryos were transferred to petri dishes with sterile E3 medium  supplemented with ampicillin 100 \u03bcg/mL  kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf  the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min  followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium  transferred to flasks and incubated at 28\u00b0C. The viability was monitored  and sterile medium was refreshed daily. At day 4  the hatched embryos were used for the sterility validation. A day 5  germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate  four replicates  i.e. eighty larvae in total  were used for each condition. post the exposure  zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly  zebrafish larvae were euthanized with 0.01% of tricaine for 5 min  collected in 1.5 mL tube  and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL  followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT  followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh  with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III  BD Biosciences  NJ  USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN  Kanther M  Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3  1862 1875 2008. 2. Bresciani E  Broadbridge E  Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae  MethodsX. 5  1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab  Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics  Pleasanton  USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly  GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads  a Master Mix containing cells  and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation  the Gel Bead was dissolved  primers containing an Illumina TruSeq Read 1read 1 sequencing primer  16 nt 10x Barcode  12 nt unique molecular identifier UMI and 30 nt polydT sequence were released  and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics  16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation  primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded  full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture  which included leftover biochemical reagents and primers. Barcoded  full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5  P7  i7 and i5 sample indexes  and TruSeq Read 2 read 2 primer sequence were added via End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 1 WT control", "SAMEA110401655", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401655|INSDC center alias:Institute of Environmental Medicine  Karolinska Institutet|INSDC center name:Institute of Environmental Medicine  Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 1 WT control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 1 WT control|sex:not available|stimulus:n1|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 1 WT control p", "Sample 1 WT control p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief  2 hpf embryos were transferred to petri dishes with sterile E3 medium  supplemented with ampicillin 100 \u03bcg/mL  kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf  the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min  followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium  transferred to flasks and incubated at 28\u00b0C. The viability was monitored  and sterile medium was refreshed daily. At day 4  the hatched embryos were used for the sterility validation. A day 5  germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate  four replicates  i.e. eighty larvae in total  were used for each condition. post the exposure  zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly  zebrafish larvae were euthanized with 0.01% of tricaine for 5 min  collected in 1.5 mL tube  and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL  followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT  followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh  with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III  BD Biosciences  NJ  USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN  Kanther M  Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3  1862 1875 2008. 2. Bresciani E  Broadbridge E  Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae  MethodsX. 5  1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab  Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics  Pleasanton  USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol.  Briefly  GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads  a Master Mix containing cells  and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation  the Gel Bead was dissolved  primers containing an Illumina TruSeq Read 1read 1 sequencing primer  16 nt 10x Barcode  12 nt unique molecular identifier UMI and 30 nt polydT sequence were released  and the co partitioned cells were lysed.  The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM  Library & Gel Bead Kit v3.1 10x Genomics  16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation  primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded  full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture  which included leftover biochemical reagents and primers. Barcoded  full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5  P7  i7 and i5 sample indexes  and TruSeq Read 2 read 2 primer sequence were added via End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:n1", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7001_S1_L002_R1_001.fastq.gz P22202_7001_S1_L002_R2_001.fastq.gz", "fastq fastq", 19063958224.0, 161558968.0, "E MTAB 11984:P22202 7001 S1 L002", "0:28 1:90", "A:5563588848;C:4022930643;G:4344549562;T:5132136743;N:752428", 28, 90, null, null, 5563588848, 4022930643, 4344549562, 5132136743, 752428, "ERX9520369", "ERS12499847", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01118, 0.89308, 0.00573, 0.23128, 0.98737, 0.79354, 0.39264, 0.54552, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11238, "ERR10782555", "ERX10233132", "ERS14439197", "ERP144048", "PRJEB58983", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E-MTAB-12503", "Transcriptome Analysis", "Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies  concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing  transport  and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here  we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function  we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns  a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling  and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent  early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b  wnt11f2  fzd7a  and vangl2. Following axis formation  rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM  that forms the hematopoietic  cardiovascular  kidney  and forelimb skeleton progenitors. Subsequently  rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a  kdrl  sox7  and the megakaryocyte regulator gfi1aa. Lastly  we document similar hematopoietic defects upon loss of vangl2. Together  our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, "Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined  pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen  with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Sample 6", "E MTAB 12503:Sample 6", null, "strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 6|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:wild type genotype|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, null, null, null, null, null, null, null, "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E MTAB 12503:Sample 6 p", "Sample 6 p", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined  pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen  with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Experimental Factor: developmental stage:bud|Experimental Factor: genotype:wild type genotype", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "RANDOM", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "ERP144048", "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", "20170530.A-6_R1.fastq.gz 20170530.A-6_R2.fastq.gz", "fastq fastq", 14809693138.0, 49038719.0, "E MTAB 12503:20170530.A 6 R", "0:151 1:151", "A:4069749497;C:3367864217;G:3430269434;T:3928390721;N:13419269", 151, 151, null, null, 4069749497, 3367864217, 3430269434, 3928390721, 13419269, "ERX10233132", "ERS14439197", "ERA20162442", "University of Zurich|European Nucleotide Archive", "University of Zurich|European Nucleotide Archive", 2, 0.83945, 0.69977, 0.27269, 0.22488, 0.74523, 0.77654, 0.4899, 0.4358, 151, 151, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "sc", "unknown", "unknown", null, "Switzerland", "2023-03-31", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11239, "ERR10782554", "ERX10233131", "ERS14439196", "ERP144048", "PRJEB58983", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E-MTAB-12503", "Transcriptome Analysis", "Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies  concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing  transport  and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here  we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function  we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns  a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling  and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent  early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b  wnt11f2  fzd7a  and vangl2. Following axis formation  rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM  that forms the hematopoietic  cardiovascular  kidney  and forelimb skeleton progenitors. Subsequently  rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a  kdrl  sox7  and the megakaryocyte regulator gfi1aa. Lastly  we document similar hematopoietic defects upon loss of vangl2. Together  our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, "Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined  pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen  with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Sample 5", "E MTAB 12503:Sample 5", null, "strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 5|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:wild type genotype|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, null, null, null, null, null, null, null, "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E MTAB 12503:Sample 5 p", "Sample 5 p", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined  pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen  with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Experimental Factor: developmental stage:bud|Experimental Factor: genotype:wild type genotype", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "RANDOM", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "ERP144048", "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", "20170530.A-5_R1.fastq.gz 20170530.A-5_R2.fastq.gz", "fastq fastq", 14954968728.0, 49519764.0, "E MTAB 12503:20170530.A 5 R", "0:151 1:151", "A:4063340262;C:3440933120;G:3455723079;T:3981396987;N:13575280", 151, 151, null, null, 4063340262, 3440933120, 3455723079, 3981396987, 13575280, "ERX10233131", "ERS14439196", "ERA20162442", "University of Zurich|European Nucleotide Archive", "University of Zurich|European Nucleotide Archive", 2, 0.85891, 0.86859, 0.28842, 0.28866, 0.7349, 0.75051, 0.45532, 0.49145, 151, 151, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "sc", "unknown", "unknown", null, "Switzerland", "2023-03-31", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11242, "ERR10782551", "ERX10233128", "ERS14439193", "ERP144048", "PRJEB58983", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E-MTAB-12503", "Transcriptome Analysis", "Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies  concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing  transport  and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here  we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function  we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns  a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling  and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent  early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b  wnt11f2  fzd7a  and vangl2. Following axis formation  rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM  that forms the hematopoietic  cardiovascular  kidney  and forelimb skeleton progenitors. Subsequently  rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a  kdrl  sox7  and the megakaryocyte regulator gfi1aa. Lastly  we document similar hematopoietic defects upon loss of vangl2. Together  our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, "Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined  pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen  with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Sample 7", "E MTAB 12503:Sample 7", null, "strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 7|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:rbm8a d5/d5|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, null, null, null, null, null, null, null, "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E MTAB 12503:Sample 7 p", "Sample 7 p", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined  pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen  with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Experimental Factor: developmental stage:bud|Experimental Factor: genotype:rbm8a d5/d5", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "RANDOM", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "ERP144048", "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", "20170530.A-7_R1.fastq.gz 20170530.A-7_R2.fastq.gz", "fastq fastq", 12726557236.0, 42140918.0, "E MTAB 12503:20170530.A 7 R", "0:151 1:151", "A:3510176970;C:2879679978;G:2932244172;T:3392911734;N:11544382", 151, 151, null, null, 3510176970, 2879679978, 2932244172, 3392911734, 11544382, "ERX10233128", "ERS14439193", "ERA20162442", "University of Zurich|European Nucleotide Archive", "University of Zurich|European Nucleotide Archive", 2, 0.88531, 0.88678, 0.33279, 0.3329, 0.73545, 0.74992, 0.52548, 0.52864, 151, 151, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "sc", "unknown", "unknown", null, "Switzerland", "2023-03-31", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11821, "ERR11834690", "ERX11232843", "ERS16254762", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 4", "SAMEA114265804", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265804|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 4|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250|organism part:whole organism|rin:9.6|sample name:E MTAB 13263:Sample 4|scientific name:Danio rerio|sex:na|stimulus:immunosuppression|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 4 p", "Sample 4 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2113_dre_C1_clobetasolpropionate_lib667285_10169_3_2.fastq.gz NG-32417_R2113_dre_C1_clobetasolpropionate_lib667285_10169_3_1.fastq.gz", "fastq fastq", 9764006696.0, 32331148.0, "E MTAB 13263:NG 32417 R2113 dre C1 clobetasolpropionate lib667285 10169 3 ", "0:151 1:151", "A:2627275848;C:2223323771;G:2323190525;T:2590103027;N:113525", 151, 151, null, null, 2627275848, 2223323771, 2323190525, 2590103027, 113525, "ERX11232843", "ERS16254762", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95424, 0.95502, 0.12596, 0.12401, 0.65884, 0.65825, 0.48134, 0.48327, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11822, "ERR11834689", "ERX11232842", "ERS16254761", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 3", "SAMEA114265803", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265803|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 3|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:0|organism part:whole organism|rin:9.4|sample name:E MTAB 13263:Sample 3|scientific name:Danio rerio|sex:na|stimulus:n1|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 3 p", "Sample 3 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2112_dre_nc_clobetasolpropionate_lib673071_10180_1_2.fastq.gz NG-32417_R2112_dre_nc_clobetasolpropionate_lib673071_10180_1_1.fastq.gz", "fastq fastq", 13326663550.0, 44128025.0, "E MTAB 13263:NG 32417 R2112 dre nc clobetasolpropionate lib673071 10180 1 ", "0:151 1:151", "A:3641640344;C:3020489123;G:3072643196;T:3591659412;N:231475", 151, 151, null, null, 3641640344, 3020489123, 3072643196, 3591659412, 231475, "ERX11232842", "ERS16254761", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95271, 0.95293, 0.11945, 0.1151, 0.65819, 0.6579, 0.47257, 0.47803, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11823, "ERR11834684", "ERX11232837", "ERS16254756", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 1", "SAMEA114265798", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265798|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 1|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:0|organism part:whole organism|rin:9.3|sample name:E MTAB 13263:Sample 1|scientific name:Danio rerio|sex:na|stimulus:n1|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 1 p", "Sample 1 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2110_dre_nc_clobetasolpropionate_lib667282_10169_3_1.fastq.gz NG-32417_R2110_dre_nc_clobetasolpropionate_lib667282_10169_3_2.fastq.gz", "fastq fastq", 10152616370.0, 33617935.0, "E MTAB 13263:NG 32417 R2110 dre nc clobetasolpropionate lib667282 10169 3 ", "0:151 1:151", "A:2743478864;C:2319134955;G:2378048842;T:2711839167;N:114542", 151, 151, null, null, 2743478864, 2319134955, 2378048842, 2711839167, 114542, "ERX11232837", "ERS16254756", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95273, 0.95457, 0.13567, 0.13174, 0.65896, 0.65841, 0.47947, 0.47815, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11824, "ERR11834691", "ERX11232844", "ERS16254763", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 5", "SAMEA114265805", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265805|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 5|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250|organism part:whole organism|rin:9.2|sample name:E MTAB 13263:Sample 5|scientific name:Danio rerio|sex:na|stimulus:immunosuppression|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 5 p", "Sample 5 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2114_dre_C1_clobetasolpropionate_lib667286_10169_3_1.fastq.gz NG-32417_R2114_dre_C1_clobetasolpropionate_lib667286_10169_3_2.fastq.gz", "fastq fastq", 11690304334.0, 38709617.0, "E MTAB 13263:NG 32417 R2114 dre C1 clobetasolpropionate lib667286 10169 3 ", "0:151 1:151", "A:3142106866;C:2677986743;G:2767050957;T:3103028799;N:130969", 151, 151, null, null, 3142106866, 2677986743, 2767050957, 3103028799, 130969, "ERX11232844", "ERS16254763", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95418, 0.95523, 0.12548, 0.12397, 0.66026, 0.66016, 0.46767, 0.47253, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11825, "ERR11834688", "ERX11232841", "ERS16254760", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 2", "SAMEA114265802", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265802|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 2|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:0|organism part:whole organism|rin:9.6|sample name:E MTAB 13263:Sample 2|scientific name:Danio rerio|sex:na|stimulus:n1|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 2 p", "Sample 2 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2111_dre_nc_clobetasolpropionate_lib673070_10180_1_1.fastq.gz NG-32417_R2111_dre_nc_clobetasolpropionate_lib673070_10180_1_2.fastq.gz", "fastq fastq", 11827163184.0, 39162792.0, "E MTAB 13263:NG 32417 R2111 dre nc clobetasolpropionate lib673070 10180 1 ", "0:151 1:151", "A:3256286612;C:2657918302;G:2701905502;T:3210846546;N:206222", 151, 151, null, null, 3256286612, 2657918302, 2701905502, 3210846546, 206222, "ERX11232841", "ERS16254760", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95203, 0.95292, 0.12277, 0.11772, 0.66048, 0.65837, 0.47243, 0.4729, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11826, "ERR11834693", "ERX11232846", "ERS16254765", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 7", "SAMEA114265807", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265807|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 7|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:4000|organism part:whole organism|rin:9.7|sample name:E MTAB 13263:Sample 7|scientific name:Danio rerio|sex:na|stimulus:immunostimulation|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 7 p", "Sample 7 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2116_dre_nc_imq_clobetasolpropionate_lib667288_10169_3_1.fastq.gz NG-32417_R2116_dre_nc_imq_clobetasolpropionate_lib667288_10169_3_2.fastq.gz", "fastq fastq", 11114382784.0, 36802592.0, "E MTAB 13263:NG 32417 R2116 dre nc imq clobetasolpropionate lib667288 10169 3 ", "0:151 1:151", "A:2997376944;C:2547742011;G:2613273908;T:2955864850;N:125071", 151, 151, null, null, 2997376944, 2547742011, 2613273908, 2955864850, 125071, "ERX11232846", "ERS16254765", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95392, 0.9539, 0.12951, 0.12707, 0.65628, 0.65731, 0.48132, 0.48179, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11827, "ERR11834695", "ERX11232848", "ERS16254767", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 9", "SAMEA114265809", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265809|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 9|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:4000|organism part:whole organism|rin:9.9|sample name:E MTAB 13263:Sample 9|scientific name:Danio rerio|sex:na|stimulus:immunostimulation|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 9 p", "Sample 9 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2118_dre_nc_imq_clobetasolpropionate_lib667290_10169_3_1.fastq.gz NG-32417_R2118_dre_nc_imq_clobetasolpropionate_lib667290_10169_3_2.fastq.gz", "fastq fastq", 9422294300.0, 31199650.0, "E MTAB 13263:NG 32417 R2118 dre nc imq clobetasolpropionate lib667290 10169 3 ", "0:151 1:151", "A:2541445212;C:2157798531;G:2214381344;T:2508560588;N:108625", 151, 151, null, null, 2541445212, 2157798531, 2214381344, 2508560588, 108625, "ERX11232848", "ERS16254767", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95337, 0.95433, 0.13046, 0.12718, 0.65845, 0.65815, 0.48198, 0.48414, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11828, "ERR11834687", "ERX11232840", "ERS16254759", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 12", "SAMEA114265801", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265801|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 12|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250 CP + 4000 IMQ|organism part:whole organism|rin:9.7|sample name:E MTAB 13263:Sample 12|scientific name:Danio rerio|sex:na|stimulus:immunosuppression and immunostimulation|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 12 p", "Sample 12 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2121_dre_C1_imq_clobetasolpropionate_lib667293_10169_3_2.fastq.gz NG-32417_R2121_dre_C1_imq_clobetasolpropionate_lib667293_10169_3_1.fastq.gz", "fastq fastq", 9285032280.0, 30745140.0, "E MTAB 13263:NG 32417 R2121 dre C1 imq clobetasolpropionate lib667293 10169 3 ", "0:151 1:151", "A:2504765552;C:2110325477;G:2196928032;T:2472905759;N:107460", 151, 151, null, null, 2504765552, 2110325477, 2196928032, 2472905759, 107460, "ERX11232840", "ERS16254759", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95411, 0.95451, 0.12502, 0.12228, 0.65829, 0.65774, 0.48259, 0.47946, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11829, "ERR11834694", "ERX11232847", "ERS16254766", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 8", "SAMEA114265808", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265808|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 8|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:4000|organism part:whole organism|rin:9.5|sample name:E MTAB 13263:Sample 8|scientific name:Danio rerio|sex:na|stimulus:immunostimulation|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 8 p", "Sample 8 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2117_dre_nc_imq_clobetasolpropionate_lib667289_10169_3_1.fastq.gz NG-32417_R2117_dre_nc_imq_clobetasolpropionate_lib667289_10169_3_2.fastq.gz", "fastq fastq", 10247527118.0, 33932209.0, "E MTAB 13263:NG 32417 R2117 dre nc imq clobetasolpropionate lib667289 10169 3 ", "0:151 1:151", "A:2766675319;C:2342307825;G:2405294253;T:2733131648;N:118073", 151, 151, null, null, 2766675319, 2342307825, 2405294253, 2733131648, 118073, "ERX11232847", "ERS16254766", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.9541, 0.95434, 0.13364, 0.13098, 0.6576, 0.6565, 0.48068, 0.48029, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11830, "ERR11834692", "ERX11232845", "ERS16254764", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 6", "SAMEA114265806", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265806|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 6|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250|organism part:whole organism|rin:10|sample name:E MTAB 13263:Sample 6|scientific name:Danio rerio|sex:na|stimulus:immunosuppression|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 6 p", "Sample 6 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2115_dre_C1_clobetasolpropionate_lib667287_10169_3_1.fastq.gz NG-32417_R2115_dre_C1_clobetasolpropionate_lib667287_10169_3_2.fastq.gz", "fastq fastq", 10440673634.0, 34571767.0, "E MTAB 13263:NG 32417 R2115 dre C1 clobetasolpropionate lib667287 10169 3 ", "0:151 1:151", "A:2805459166;C:2397120759;G:2463616298;T:2774359989;N:117422", 151, 151, null, null, 2805459166, 2397120759, 2463616298, 2774359989, 117422, "ERX11232845", "ERS16254764", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95568, 0.9548, 0.13056, 0.12563, 0.65882, 0.66332, 0.49162, 0.48586, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11831, "ERR11834685", "ERX11232838", "ERS16254757", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 10", "SAMEA114265799", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265799|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 10|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250 CP + 4000 IMQ|organism part:whole organism|rin:9.4|sample name:E MTAB 13263:Sample 10|scientific name:Danio rerio|sex:na|stimulus:immunosuppression and immunostimulation|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 10 p", "Sample 10 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2119_dre_C1_imq_clobetasolpropionate_lib667291_10169_3_2.fastq.gz NG-32417_R2119_dre_C1_imq_clobetasolpropionate_lib667291_10169_3_1.fastq.gz", "fastq fastq", 11267068246.0, 37308173.0, "E MTAB 13263:NG 32417 R2119 dre C1 imq clobetasolpropionate lib667291 10169 3 ", "0:151 1:151", "A:3042432765;C:2574394239;G:2642086000;T:3008026288;N:128954", 151, 151, null, null, 3042432765, 2574394239, 2642086000, 3008026288, 128954, "ERX11232838", "ERS16254757", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.95345, 0.95411, 0.13324, 0.13062, 0.65796, 0.65892, 0.48176, 0.48174, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [11832, "ERR11834686", "ERX11232839", "ERS16254758", "ERP150242", "PRJEB65099", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E-MTAB-13263", "Transcriptome Analysis", "Within the regulatory framework on the approval of new substances  the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances  clobetasol propionate CP  CAS 25122 46 7 and imiquimod IMQ  CAS 99011 02 6  on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs  ii identify compound specific molecular signatures  iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this  the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge  i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test OECD 236  15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf  all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode  producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", null, "Protocols: At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", "Sample 11", "SAMEA114265800", "Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany", "ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265800|INSDC center name:Department Ecotoxicogenomics  Fraunhofer Institute for Molecular Biology and Applied Ecology IME  Schmallenberg  Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 11|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250 CP + 4000 IMQ|organism part:whole organism|rin:10|sample name:E MTAB 13263:Sample 11|scientific name:Danio rerio|sex:na|stimulus:immunosuppression and immunostimulation|strain:Wildtype AB", null, null, null, null, null, null, null, null, "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "E MTAB 13263:Sample 11 p", "Sample 11 p", "Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "At 72 hpf  for each sample  all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos.   For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the \u201cGrowth protocol\u201d. For detailed information about exposure duration and conditions please refer to the \u201cTreatment protocol\u201d.  Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH  Germany  and were kept in 150 l tanks under flow through conditions at 26 \u00b1 2 \u00b0C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME  Schmallenberg  Germany. TetraMin\u00ae Tetra Werke  Germany was used as daily main feed  regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks  making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection  glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD  2013. The morning post  eggs were collected from the trays using a sieve  rinsed with pre tempered system water to remove remaining feces  gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 \u00b1 1 \u00b0C until further use.  Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see \u201ctreatment protocol\u201d   Clobetasol propionate 21 Chlor 9 fluor 11\u03b2 17 dihydroxy 16\u03b2 methylpregna 1 4 dien 3 20 dion 17 propionat  purity \u2265 98 %  CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine  purity \u2265 95 %  CAS no. 9901 02 6 were purchased from BIOMOL GmbH  Hamburg  Germany. For IMQ  a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP  a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ  2242.5 \u00b5l of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions  equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP  20 \u00b5l of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 \u00b5l DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H  Bandelin  Germany. Final solutions were thus cu red water with 0.01 % DMSO  4666 nM IMQ in cu red water with 0.01 % DMSO  250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions.  The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment.  In order to minimize the differences between replicates and conditions in exposure durations  the selection of embryos was performed in two steps: at 2 hpf  approx. 50 embryos from spawning batches with a minimum fertilization rate \u2265 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound  embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution  15 healthy and fertilized embryos  indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 \u00b1 1\u00b0C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn  Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline  namely coagulation  lack of somite formation  lack of a heartbeat  non detachment of the tail OECD  2013 as well as further indicators of malformations such as scoliosis  formation of edema or lack of pigmentation. At 47 hpf  embryos were manually dechorionated using two pointy forceps. At 48 hpf  the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions  embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes  filled with 6 ml of the corresponding fresh medium. Due to this dilution  a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution.  RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel  D\u00fcren  Germany. Briefly  frozen embryos were transferred to screw cap tubes filled with 350 \u00b5l kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals  Irvine  USA and homogenized using a FastPrep 24 MP Biomedicals  Irvine  USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures  purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific  Waltham  USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent  Santa Clara  USA with samples prepared using a RNA Pico 6000 kit Agilent  Santa Clara  USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at  80\u00b0C until they were send to the sequencing facility on dry ice.  High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing  performed by Eurofins Genomics Germany GmbH  Ebersberg  Germany. Briefly  Poly A containing protein coding mRNA molecules were purified from total RNA  randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.", null, "RNA-Seq", "TRANSCRIPTOMIC", "PolyA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP150242", "Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of  TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression", "ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01", "NG-32417_R2120_dre_C1_imq_clobetasolpropionate_lib667292_10169_3_2.fastq.gz NG-32417_R2120_dre_C1_imq_clobetasolpropionate_lib667292_10169_3_1.fastq.gz", "fastq fastq", 9922486330.0, 32855915.0, "E MTAB 13263:NG 32417 R2120 dre C1 imq clobetasolpropionate lib667292 10169 3 ", "0:151 1:151", "A:2673462020;C:2268840894;G:2345658284;T:2634411815;N:113317", 151, 151, null, null, 2673462020, 2268840894, 2345658284, 2634411815, 113317, "ERX11232839", "ERS16254758", "ERA26742386", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.9551, 0.95621, 0.1193, 0.11675, 0.65833, 0.65827, 0.48596, 0.48547, 151, 151, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "unknown", "poly_a", "trueseq", "bulk", "unknown", "unknown", null, "United Kingdom", "2023-12-01", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"], [13299, "ERR984502", "ERX1065723", "ERS715232", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367580", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367580|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 24 sc 2286404|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCAGCTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 24 sc 2286404|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#24", "13741631", "Illumina sequencing of library 13741631  constructed from sample accession ERS715232 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TTCAGCTC.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#24.cram", "cram", 570705070.0, 4390039.0, "SC RUN 16164 8#24", "0:55 1:75", "A:167603013;C:88769000;G:138595187;T:175336986;N:400884", 55, 75, null, null, 167603013, 88769000, 138595187, 175336986, 400884, "ERX1065723", "ERS715232", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.18131, 0.44012, 0.12372, 0.1122, 0.96759, 0.93835, 0.63426, 0.57651, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13300, "ERR984501", "ERX1065722", "ERS715231", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367579", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367579|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 23 sc 2286403|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACTAGTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 23 sc 2286403|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#23", "13741630", "Illumina sequencing of library 13741630  constructed from sample accession ERS715231 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TACTAGTC.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#23.cram", "cram", 682831890.0, 5252553.0, "SC RUN 16164 8#23", "0:55 1:75", "A:201267036;C:106752351;G:152982998;T:221347859;N:481646", 55, 75, null, null, 201267036, 106752351, 152982998, 221347859, 481646, "ERX1065722", "ERS715231", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.19992, 0.5315, 0.13001, 0.13817, 0.96025, 0.92261, 0.60527, 0.55787, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13301, "ERR984500", "ERX1065721", "ERS715230", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367578", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367578|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 22 sc 2286402|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCAGATTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 22 sc 2286402|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#22", "13741629", "Illumina sequencing of library 13741629  constructed from sample accession ERS715230 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCAGATTC.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#22.cram", "cram", 822730220.0, 6328694.0, "SC RUN 16164 8#22", "0:55 1:75", "A:241693794;C:130571512;G:177853465;T:272028770;N:582679", 55, 75, null, null, 241693794, 130571512, 177853465, 272028770, 582679, "ERX1065721", "ERS715230", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.2212, 0.54203, 0.15635, 0.13659, 0.96159, 0.9221, 0.59031, 0.55109, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13302, "ERR984499", "ERX1065720", "ERS715229", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367577", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367577|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 21 sc 2286401|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TATGCCAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 21 sc 2286401|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#21", "13741628", "Illumina sequencing of library 13741628  constructed from sample accession ERS715229 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TATGCCAG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#21.cram", "cram", 616663190.0, 4743563.0, "SC RUN 16164 8#21", "0:55 1:75", "A:176848806;C:99629161;G:137029831;T:202730142;N:425250", 55, 75, null, null, 176848806, 99629161, 137029831, 202730142, 425250, "ERX1065720", "ERS715229", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21864, 0.57174, 0.1516, 0.14103, 0.95905, 0.9192, 0.55513, 0.51633, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13303, "ERR984498", "ERX1065719", "ERS715228", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367576", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367576|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 20 sc 2286400|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGGCTCAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 20 sc 2286400|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#20", "13741627", "Illumina sequencing of library 13741627  constructed from sample accession ERS715228 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGGCTCAG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#20.cram", "cram", 708059950.0, 5446615.0, "SC RUN 16164 8#20", "0:55 1:75", "A:195264246;C:129715499;G:160155422;T:222438911;N:485872", 55, 75, null, null, 195264246, 129715499, 160155422, 222438911, 485872, "ERX1065719", "ERS715228", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.19642, 0.48584, 0.11921, 0.11811, 0.95422, 0.91843, 0.62494, 0.56646, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13304, "ERR984497", "ERX1065718", "ERS715227", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367575", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367575|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 19 sc 2286399|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCATTGAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 19 sc 2286399|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#19", "13741626", "Illumina sequencing of library 13741626  constructed from sample accession ERS715227 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCATTGAG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#19.cram", "cram", 651943890.0, 5014953.0, "SC RUN 16164 8#19", "0:55 1:75", "A:191809575;C:111573276;G:144109125;T:204007845;N:444069", 55, 75, null, null, 191809575, 111573276, 144109125, 204007845, 444069, "ERX1065718", "ERS715227", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.17909, 0.44616, 0.12326, 0.12424, 0.96256, 0.93432, 0.56606, 0.52819, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13305, "ERR984496", "ERX1065717", "ERS715226", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367574", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367574|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 18 sc 2286398|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGTATGCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 18 sc 2286398|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#18", "13741625", "Illumina sequencing of library 13741625  constructed from sample accession ERS715226 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGTATGCG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#18.cram", "cram", 610476620.0, 4695974.0, "SC RUN 16164 8#18", "0:55 1:75", "A:181332724;C:99472616;G:132687929;T:196571397;N:411954", 55, 75, null, null, 181332724, 99472616, 132687929, 196571397, 411954, "ERX1065717", "ERS715226", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.22636, 0.55559, 0.16123, 0.16819, 0.96025, 0.92967, 0.59576, 0.54685, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13306, "ERR984495", "ERX1065716", "ERS715225", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367573", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367573|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 17 sc 2286397|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCCAGTCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 17 sc 2286397|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#17", "13741624", "Illumina sequencing of library 13741624  constructed from sample accession ERS715225 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCCAGTCG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#17.cram", "cram", 712320830.0, 5479391.0, "SC RUN 16164 8#17", "0:55 1:75", "A:204316194;C:115198795;G:158608369;T:233717227;N:480245", 55, 75, null, null, 204316194, 115198795, 158608369, 233717227, 480245, "ERX1065716", "ERS715225", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.2492, 0.59739, 0.17155, 0.14862, 0.95657, 0.92056, 0.58039, 0.53475, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13307, "ERR984494", "ERX1065715", "ERS715224", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367572", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367572|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 16 sc 2286396|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAAGTTCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 16 sc 2286396|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#16", "13741623", "Illumina sequencing of library 13741623  constructed from sample accession ERS715224 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TAAGTTCG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#16.cram", "cram", 793769340.0, 6105918.0, "SC RUN 16164 8#16", "0:55 1:75", "A:232264866;C:130235228;G:171709756;T:259013732;N:545758", 55, 75, null, null, 232264866, 130235228, 171709756, 259013732, 545758, "ERX1065715", "ERS715224", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.22065, 0.5061, 0.15274, 0.13242, 0.95779, 0.92575, 0.57619, 0.53869, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13308, "ERR984493", "ERX1065714", "ERS715223", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367571", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367571|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 15 sc 2286395|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCAGGAGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 15 sc 2286395|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#15", "13741622", "Illumina sequencing of library 13741622  constructed from sample accession ERS715223 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCAGGAGG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#15.cram", "cram", 785479760.0, 6042152.0, "SC RUN 16164 8#15", "0:55 1:75", "A:221631721;C:150326010;G:172964474;T:240036379;N:521176", 55, 75, null, null, 221631721, 150326010, 172964474, 240036379, 521176, "ERX1065714", "ERS715223", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.26761, 0.37751, 0.19858, 0.09301, 0.95162, 0.93129, 0.56282, 0.54289, 55, 75, "B", "B", "mate1-mate2 similar by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13309, "ERR984492", "ERX1065713", "ERS715222", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367570", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367570|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 14 sc 2286394|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCTCACGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 14 sc 2286394|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#14", "13741621", "Illumina sequencing of library 13741621  constructed from sample accession ERS715222 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCTCACGG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#14.cram", "cram", 673293530.0, 5179181.0, "SC RUN 16164 8#14", "0:55 1:75", "A:188203068;C:115934699;G:155324541;T:213374769;N:456453", 55, 75, null, null, 188203068, 115934699, 155324541, 213374769, 456453, "ERX1065713", "ERS715222", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21855, 0.57586, 0.15197, 0.14501, 0.95946, 0.93077, 0.56453, 0.52351, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13310, "ERR984491", "ERX1065712", "ERS715221", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367569", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367569|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 13 sc 2286393|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACTTCGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 13 sc 2286393|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#13", "13741620", "Illumina sequencing of library 13741620  constructed from sample accession ERS715221 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TACTTCGG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#13.cram", "cram", 783634020.0, 6027954.0, "SC RUN 16164 8#13", "0:55 1:75", "A:231435775;C:129417634;G:178939153;T:243302897;N:538561", 55, 75, null, null, 231435775, 129417634, 178939153, 243302897, 538561, "ERX1065712", "ERS715221", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.20287, 0.52947, 0.14655, 0.13479, 0.96299, 0.93703, 0.56376, 0.52626, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13311, "ERR984490", "ERX1065711", "ERS715220", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367568", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367568|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 12 sc 2286392|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGAACTGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 12 sc 2286392|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#12", "13741619", "Illumina sequencing of library 13741619  constructed from sample accession ERS715220 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGAACTGG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#12.cram", "cram", 746397730.0, 5741521.0, "SC RUN 16164 8#12", "0:55 1:75", "A:217959769;C:123138989;G:158254272;T:246531930;N:512770", 55, 75, null, null, 217959769, 123138989, 158254272, 246531930, 512770, "ERX1065711", "ERS715220", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.20695, 0.57674, 0.14123, 0.14978, 0.95899, 0.9138, 0.56705, 0.57522, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13312, "ERR984489", "ERX1065710", "ERS715219", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367567", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367567|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 11 sc 2286391|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTGGTATG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 11 sc 2286391|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#11", "13741618", "Illumina sequencing of library 13741618  constructed from sample accession ERS715219 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TTGGTATG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#11.cram", "cram", 630077890.0, 4846753.0, "SC RUN 16164 8#11", "0:55 1:75", "A:181555459;C:107913967;G:131155798;T:209016157;N:436509", 55, 75, null, null, 181555459, 107913967, 131155798, 209016157, 436509, "ERX1065710", "ERS715219", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.23034, 0.58664, 0.14994, 0.16031, 0.9517, 0.9134, 0.43491, 0.55537, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13313, "ERR984488", "ERX1065709", "ERS715218", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367566", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:57Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367566|INSDC center name:SC|INSDC first public:2015 08 18T09:04:57Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 10 sc 2286390|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAACGCTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 10 sc 2286390|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#10", "13741617", "Illumina sequencing of library 13741617  constructed from sample accession ERS715218 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TAACGCTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#10.cram", "cram", 522425410.0, 4018657.0, "SC RUN 16164 8#10", "0:55 1:75", "A:136789388;C:88820863;G:131305976;T:165156817;N:352366", 55, 75, null, null, 136789388, 88820863, 131305976, 165156817, 352366, "ERX1065709", "ERS715218", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.16976, 0.61187, 0.10677, 0.16777, 0.96096, 0.93093, 0.59603, 0.58893, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13314, "ERR984487", "ERX1065708", "ERS715217", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367565", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367565|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 9 sc 2286389|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCGAAGTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 9 sc 2286389|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#9", "13741616", "Illumina sequencing of library 13741616  constructed from sample accession ERS715217 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCGAAGTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#9.cram", "cram", 662372100.0, 5095170.0, "SC RUN 16164 8#9", "0:55 1:75", "A:186346528;C:114321445;G:137139385;T:224106023;N:458719", 55, 75, null, null, 186346528, 114321445, 137139385, 224106023, 458719, "ERX1065708", "ERS715217", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.22854, 0.63729, 0.14734, 0.16003, 0.95089, 0.90751, 0.51773, 0.50977, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13315, "ERR984486", "ERX1065707", "ERS715216", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367564", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367564|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 8 sc 2286388|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCCATTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 8 sc 2286388|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#8", "13741615", "Illumina sequencing of library 13741615  constructed from sample accession ERS715216 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TTCCATTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#8.cram", "cram", 857007060.0, 6592362.0, "SC RUN 16164 8#8", "0:55 1:75", "A:257958701;C:131661477;G:190276240;T:276500331;N:610311", 55, 75, null, null, 257958701, 131661477, 190276240, 276500331, 610311, "ERX1065707", "ERS715216", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21266, 0.595, 0.14225, 0.14037, 0.95964, 0.92232, 0.54595, 0.53111, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13316, "ERR984485", "ERX1065706", "ERS715215", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367563", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367563|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 7 sc 2286387|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAGTCTTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 7 sc 2286387|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#7", "13741614", "Illumina sequencing of library 13741614  constructed from sample accession ERS715215 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TAGTCTTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#7.cram", "cram", 742942590.0, 5714943.0, "SC RUN 16164 8#7", "0:55 1:75", "A:228799118;C:117009382;G:166056205;T:230565934;N:511951", 55, 75, null, null, 228799118, 117009382, 166056205, 230565934, 511951, "ERX1065706", "ERS715215", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.19264, 0.50767, 0.1428, 0.15173, 0.9666, 0.94606, 0.57413, 0.53902, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13317, "ERR984484", "ERX1065705", "ERS715214", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367562", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367562|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 6 sc 2286386|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGTGGTTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 6 sc 2286386|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#6", "13741613", "Illumina sequencing of library 13741613  constructed from sample accession ERS715214 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGTGGTTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#6.cram", "cram", 908951420.0, 6991934.0, "SC RUN 16164 8#6", "0:55 1:75", "A:266807632;C:158160652;G:194742499;T:288619707;N:620930", 55, 75, null, null, 266807632, 158160652, 194742499, 288619707, 620930, "ERX1065705", "ERS715214", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.20021, 0.52069, 0.13208, 0.1197, 0.95775, 0.93324, 0.52946, 0.52456, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13318, "ERR984483", "ERX1065704", "ERS715213", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367561", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367561|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 5 sc 2286385|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCCTCAAT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 5 sc 2286385|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#5", "13741612", "Illumina sequencing of library 13741612  constructed from sample accession ERS715213 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCCTCAAT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#5.cram", "cram", 771659330.0, 5935841.0, "SC RUN 16164 8#5", "0:55 1:75", "A:225161011;C:121434126;G:174639826;T:249876341;N:548026", 55, 75, null, null, 225161011, 121434126, 174639826, 249876341, 548026, "ERX1065704", "ERS715213", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21069, 0.57513, 0.14228, 0.12501, 0.95672, 0.92488, 0.54397, 0.52111, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13319, "ERR984482", "ERX1065703", "ERS715212", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367560", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367560|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 4 sc 2286384|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACAGGAT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 4 sc 2286384|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#4", "13741611", "Illumina sequencing of library 13741611  constructed from sample accession ERS715212 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TACAGGAT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#4.cram", "cram", 784183790.0, 6032183.0, "SC RUN 16164 8#4", "0:55 1:75", "A:219327080;C:128313123;G:169475262;T:266538585;N:529740", 55, 75, null, null, 219327080, 128313123, 169475262, 266538585, 529740, "ERX1065703", "ERS715212", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.20392, 0.58702, 0.12804, 0.12156, 0.96047, 0.9133, 0.61438, 0.53735, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13320, "ERR984481", "ERX1065702", "ERS715211", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367559", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367559|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 3 sc 2286383|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAGTGACT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 3 sc 2286383|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#3", "13741610", "Illumina sequencing of library 13741610  constructed from sample accession ERS715211 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TAGTGACT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#3.cram", "cram", 810265170.0, 6232809.0, "SC RUN 16164 8#3", "0:55 1:75", "A:237117705;C:130774361;G:170817877;T:270997878;N:557349", 55, 75, null, null, 237117705, 130774361, 170817877, 270997878, 557349, "ERX1065702", "ERS715211", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.22414, 0.5931, 0.14561, 0.13304, 0.95651, 0.91423, 0.59465, 0.54152, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13321, "ERR984480", "ERX1065701", "ERS715210", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367558", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367558|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 2 sc 2286382|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCCTGCT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 2 sc 2286382|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#2", "13741609", "Illumina sequencing of library 13741609  constructed from sample accession ERS715210 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TTCCTGCT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#2.cram", "cram", 698194770.0, 5370729.0, "SC RUN 16164 8#2", "0:55 1:75", "A:190784194;C:110138446;G:188418516;T:208362798;N:490816", 55, 75, null, null, 190784194, 110138446, 188418516, 208362798, 490816, "ERX1065701", "ERS715210", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.17398, 0.58306, 0.10399, 0.12476, 0.96372, 0.94468, 0.64309, 0.54721, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13322, "ERR984479", "ERX1065700", "ERS715209", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367557", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367557|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 1 sc 2286381|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGCGATCT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 1 sc 2286381|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#1", "13741608", "Illumina sequencing of library 13741608  constructed from sample accession ERS715209 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGCGATCT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#1.cram", "cram", 882032060.0, 6784862.0, "SC RUN 16164 8#1", "0:55 1:75", "A:258098215;C:144707482;G:194402683;T:284218124;N:605556", 55, 75, null, null, 258098215, 144707482, 194402683, 284218124, 605556, "ERX1065700", "ERS715209", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21585, 0.55375, 0.14409, 0.13461, 0.96015, 0.9246, 0.61791, 0.5495, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [24967, "SRR25557924", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5_S3_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L001_R2_001.fastq.gz", "fastq fastq fastq", 783833459.0, 6171917.0, "GSM7688796 r1", "0:8 1:28 2:91", "A:165863131;C:117191802;G:128146802;T:150173329;N:269383", 8, 28, 91, null, 165863131, 117191802, 128146802, 150173329, 269383, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89127, null, 0.221, null, 0.78317, null, 0.52195, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24968, "SRR25557925", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-3_S2_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L002_R2_001.fastq.gz", "fastq fastq fastq", 7208342327.0, 56758601.0, "GSM7688796 r10", "0:8 1:28 2:91", "A:1518008610;C:1083228714;G:1190089577;T:1369772782;N:3933008", 8, 28, 91, null, 1518008610, 1083228714, 1190089577, 1369772782, 3933008, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.88938, null, 0.21991, null, 0.79088, null, 0.53044, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24969, "SRR25557926", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-3_S2_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L003_R2_001.fastq.gz", "fastq fastq fastq", 7337435414.0, 57775082.0, "GSM7688796 r11", "0:8 1:28 2:91", "A:1545051667;C:1103394058;G:1211577972;T:1395133564;N:2375201", 8, 28, 91, null, 1545051667, 1103394058, 1211577972, 1395133564, 2375201, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.88966, null, 0.2211, null, 0.78961, null, 0.53156, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24970, "SRR25557927", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-3_S2_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L004_R2_001.fastq.gz", "fastq fastq fastq", 7218240453.0, 56836539.0, "GSM7688796 r12", "0:8 1:28 2:91", "A:1521442237;C:1084242714;G:1191469584;T:1372277539;N:2692975", 8, 28, 91, null, 1521442237, 1084242714, 1191469584, 1372277539, 2692975, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.8888, null, 0.2174, null, 0.79172, null, 0.53311, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24971, "SRR25557928", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5_S3_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L002_R2_001.fastq.gz", "fastq fastq fastq", 774302109.0, 6096867.0, "GSM7688796 r2", "0:8 1:28 2:91", "A:163989105;C:115793536;G:126358958;T:148430473;N:242825", 8, 28, 91, null, 163989105, 115793536, 126358958, 148430473, 242825, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89371, null, 0.2215, null, 0.78253, null, 0.53282, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24972, "SRR25557929", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5_S3_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L003_R2_001.fastq.gz", "fastq fastq fastq", 790430093.0, 6223859.0, "GSM7688796 r3", "0:8 1:28 2:91", "A:167465790;C:118269595;G:129024496;T:151447225;N:164063", 8, 28, 91, null, 167465790, 118269595, 129024496, 151447225, 164063, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89284, null, 0.21955, null, 0.78356, null, 0.53369, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24973, "SRR25557930", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5_S3_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L004_R2_001.fastq.gz", "fastq fastq fastq", 778936974.0, 6133362.0, "GSM7688796 r4", "0:8 1:28 2:91", "A:165013558;C:116498474;G:127146290;T:149314806;N:162814", 8, 28, 91, null, 165013558, 116498474, 127146290, 149314806, 162814, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89195, null, 0.2209, null, 0.78196, null, 0.53121, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24974, "SRR25557931", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-2_S2_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L001_R2_001.fastq.gz", "fastq fastq fastq", 4174244890.0, 32868070.0, "GSM7688796 r5", "0:8 1:28 2:91", "A:882060228;C:627098568;G:684890393;T:795503862;N:1441319", 8, 28, 91, null, 882060228, 627098568, 684890393, 795503862, 1441319, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89137, null, 0.22041, null, 0.77926, null, 0.52991, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24975, "SRR25557932", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-2_S2_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L002_R2_001.fastq.gz", "fastq fastq fastq", 4150210902.0, 32678826.0, "GSM7688796 r6", "0:8 1:28 2:91", "A:878218072;C:623662333;G:679977758;T:790589138;N:1325865", 8, 28, 91, null, 878218072, 623662333, 679977758, 790589138, 1325865, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89264, null, 0.22159, null, 0.78121, null, 0.52989, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24976, "SRR25557933", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-2_S2_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L003_R2_001.fastq.gz", "fastq fastq fastq", 4198043547.0, 33055461.0, "GSM7688796 r7", "0:8 1:28 2:91", "A:888287475;C:630914324;G:688006958;T:799794077;N:1044117", 8, 28, 91, null, 888287475, 630914324, 688006958, 799794077, 1044117, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.8912, null, 0.21889, null, 0.7822, null, 0.52774, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24977, "SRR25557934", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-2_S2_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L004_R2_001.fastq.gz", "fastq fastq fastq", 4154784553.0, 32714839.0, "GSM7688796 r8", "0:8 1:28 2:91", "A:880520885;C:624179045;G:680076172;T:791297938;N:976309", 8, 28, 91, null, 880520885, 624179045, 680076172, 791297938, 976309, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.8927, null, 0.22063, null, 0.78216, null, 0.5346, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24978, "SRR25557935", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-3_S2_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L001_R2_001.fastq.gz", "fastq fastq fastq", 7231973725.0, 56944675.0, "GSM7688796 r9", "0:8 1:28 2:91", "A:1522035629;C:1085783001;G:1195535350;T:1374885853;N:3725592", 8, 28, 91, null, 1522035629, 1085783001, 1195535350, 1374885853, 3725592, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.88833, null, 0.22076, null, 0.79056, null, 0.52744, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24979, "SRR25557936", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8_S2_L001_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L001_R2_001.fastq.gz", "fastq fastq fastq", 699161670.0, 5505210.0, "GSM7688795 r1", "0:8 1:28 2:91", "A:149927051;C:100829317;G:116075103;T:133908276;N:234363", 8, 28, 91, null, 149927051, 100829317, 116075103, 133908276, 234363, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85081, null, 0.21257, null, 0.80426, null, 0.52779, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24980, "SRR25557937", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-3_S1_L002_I1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L002_R2_001.fastq.gz", "fastq fastq fastq", 8174836330.0, 64368790.0, "GSM7688795 r10", "0:8 1:28 2:91", "A:1748532358;C:1187854561;G:1356279755;T:1560491610;N:4401606", 8, 28, 91, null, 1748532358, 1187854561, 1356279755, 1560491610, 4401606, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85069, null, 0.20989, null, 0.81049, null, 0.52069, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24981, "SRR25557938", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-3_S1_L003_I1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L003_R2_001.fastq.gz", "fastq fastq fastq", 8307872640.0, 65416320.0, "GSM7688795 r11", "0:8 1:28 2:91", "A:1778112295;C:1207569588;G:1377139686;T:1587359299;N:2704252", 8, 28, 91, null, 1778112295, 1207569588, 1377139686, 1587359299, 2704252, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85151, null, 0.21054, null, 0.80813, null, 0.52675, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24982, "SRR25557939", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-3_S1_L004_R2_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L004_I1_001.fastq.gz", "fastq fastq fastq", 8197523483.0, 64547429.0, "GSM7688795 r12", "0:8 1:28 2:91", "A:1755321680;C:1190415496;G:1359502433;T:1565500310;N:3076120", 8, 28, 91, null, 1755321680, 1190415496, 1359502433, 1565500310, 3076120, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.84956, null, 0.20968, null, 0.81113, null, 0.52382, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24983, "SRR25557940", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8_S2_L002_R2_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L002_I1_001.fastq.gz", "fastq fastq fastq", 690198899.0, 5434637.0, "GSM7688795 r2", "0:8 1:28 2:91", "A:147896108;C:99606794;G:114681063;T:132140792;N:227210", 8, 28, 91, null, 147896108, 99606794, 114681063, 132140792, 227210, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85228, null, 0.21261, null, 0.803, null, 0.52382, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24984, "SRR25557941", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8_S2_L003_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L003_R2_001.fastq.gz", "fastq fastq fastq", 702239515.0, 5529445.0, "GSM7688795 r3", "0:8 1:28 2:91", "A:150477574;C:101266550;G:116878466;T:134415299;N:141606", 8, 28, 91, null, 150477574, 101266550, 116878466, 134415299, 141606, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85364, null, 0.21344, null, 0.80346, null, 0.52158, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24985, "SRR25557942", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8_S2_L004_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L004_R2_001.fastq.gz", "fastq fastq fastq", 694537346.0, 5468798.0, "GSM7688795 r4", "0:8 1:28 2:91", "A:148876263;C:100202441;G:115455212;T:132982029;N:144673", 8, 28, 91, null, 148876263, 100202441, 115455212, 132982029, 144673, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85238, null, 0.2138, null, 0.80379, null, 0.52691, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24986, "SRR25557943", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-2_S1_L001_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L001_I1_001.fastq.gz", "fastq fastq fastq", 4324997954.0, 34055102.0, "GSM7688795 r5", "0:8 1:28 2:91", "A:924471384;C:626141120;G:722856562;T:824050239;N:1494977", 8, 28, 91, null, 924471384, 626141120, 722856562, 824050239, 1494977, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85083, null, 0.21122, null, 0.80472, null, 0.524, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24987, "SRR25557944", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-2_S1_L002_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L002_I1_001.fastq.gz", "fastq fastq fastq", 4306232942.0, 33907346.0, "GSM7688795 r6", "0:8 1:28 2:91", "A:920615353;C:623090513;G:721201055;T:819291149;N:1370416", 8, 28, 91, null, 920615353, 623090513, 721201055, 819291149, 1370416, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85335, null, 0.21315, null, 0.80206, null, 0.51508, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24988, "SRR25557945", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-2_S1_L003_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L003_I1_001.fastq.gz", "fastq fastq fastq", 4348436312.0, 34239656.0, "GSM7688795 r7", "0:8 1:28 2:91", "A:930204208;C:629005094;G:727756813;T:827747478;N:1095103", 8, 28, 91, null, 930204208, 629005094, 727756813, 827747478, 1095103, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85369, null, 0.21216, null, 0.80503, null, 0.52219, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24989, "SRR25557946", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-2_S1_L004_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L004_I1_001.fastq.gz", "fastq fastq fastq", 4313915172.0, 33967836.0, "GSM7688795 r8", "0:8 1:28 2:91", "A:922742754;C:623127831;G:724345885;T:819819593;N:1037013", 8, 28, 91, null, 922742754, 623127831, 724345885, 819819593, 1037013, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85456, null, 0.21236, null, 0.80223, null, 0.5216, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24990, "SRR25557947", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "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: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 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 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m 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 \u00b5g/ml and 5 \u00b5M 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 \u00b5m 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 \u00b5m 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.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-3_S1_L001_R2_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L001_I1_001.fastq.gz", "fastq fastq fastq", 8184961024.0, 64448512.0, "GSM7688795 r9", "0:8 1:28 2:91", "A:1748819280;C:1187802244;G:1360994184;T:1562975470;N:4223414", 8, 28, 91, null, 1748819280, 1187802244, 1360994184, 1562975470, 4223414, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.8493, null, 0.21072, null, 0.80967, null, 0.52593, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [25182, "SRR25670729", "SRX21396042", "SRS18636200", "SRP455680", "PRJNA1006406", "Control of polyA tail length and translation in vertebrate oocytes and early embryos", "GSE241107", "Other", "During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.", null, null, null, "Fish embryo mRNA germ ring PAL seq v4", "GSM7716871", null, "source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing", "Fish embryo mRNA germ ring PAL seq v4", "For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters \u201c m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7\u201d. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters \u201c  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate\u201d. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4\u20137 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4", "embryo", null, "Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37\u00b0C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3\u02b9 adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer\u2019s suggested protocol for 10\u201315 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3\u02b9 adapter KXS013 was used for 3\u02b9 end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "tissue:embryo|treatment:N1", "GSM7716871", "GSM7716871: Fish embryo mRNA germ ring PAL seq v4; Danio rerio; OTHER", "GSM7716871 r1", "GSM7716871", "1", "Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37\u00b0C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3\u02b9 adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10\u201315 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3\u02b9 adapter KXS013 was used for 3\u02b9 end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "SRP455680", null, null, "Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep1_raw_read2.fastq.gz Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep1_raw_read1.fastq.gz", "fastq fastq", 2834842912.0, 9234016.0, "GSM7716871 r1", "0:52 1:255", "A:725811118;C:702410592;G:747583409;T:651018348;N:8019445", 52, 255, null, null, 725811118, 702410592, 747583409, 651018348, 8019445, "SRX21396042", "SRS18636200", "SRA1694849", "Whitehead Institute", "Whitehead Institute", 2, 0.00023, 0.30494, 8e-05, 0.01793, 0.99967, 0.99971, 0.5, 1.0, 52, 255, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "bulk", "bulk", null, "United States", "2023-08-17", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [25183, "SRR25670730", "SRX21396042", "SRS18636200", "SRP455680", "PRJNA1006406", "Control of polyA tail length and translation in vertebrate oocytes and early embryos", "GSE241107", "Other", "During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.", null, null, null, "Fish embryo mRNA germ ring PAL seq v4", "GSM7716871", null, "source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing", "Fish embryo mRNA germ ring PAL seq v4", "For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters \u201c m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7\u201d. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters \u201c  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate\u201d. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4\u20137 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4", "embryo", null, "Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37\u00b0C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3\u02b9 adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer\u2019s suggested protocol for 10\u201315 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3\u02b9 adapter KXS013 was used for 3\u02b9 end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "tissue:embryo|treatment:N1", "GSM7716871", "GSM7716871: Fish embryo mRNA germ ring PAL seq v4; Danio rerio; OTHER", "GSM7716871 r1", "GSM7716871", "1", "Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37\u00b0C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3\u02b9 adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10\u201315 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3\u02b9 adapter KXS013 was used for 3\u02b9 end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "SRP455680", null, null, "Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep2_raw_read2.fastq.gz", "fastq fastq", 3473033898.0, 11312814.0, "GSM7716871 r2", "0:52 1:255", "A:853178471;C:899976159;G:962100712;T:750811461;N:6967095", 52, 255, null, null, 853178471, 899976159, 962100712, 750811461, 6967095, "SRX21396042", "SRS18636200", "SRA1694849", "Whitehead Institute", "Whitehead Institute", 2, 0.00049, 0.0, 0.00012, 0.0, 0.99941, 1.0, 0.64864, null, 52, 255, "T", "T", "mates < 9% mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "bulk", "bulk", null, "United States", "2023-08-17", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [25184, "SRR25670731", "SRX21396041", "SRS18636199", "SRP455680", "PRJNA1006406", "Control of polyA tail length and translation in vertebrate oocytes and early embryos", "GSE241107", "Other", "During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.", null, null, null, "Fish embryo mRNA zfs:0000015 PAL seq v4", "GSM7716870", null, "source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing", "Fish embryo mRNA zfs:0000015 PAL seq v4", "For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters \u201c m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7\u201d. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters \u201c  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate\u201d. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4\u20137 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4", "embryo", null, "Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37\u00b0C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3\u02b9 adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer\u2019s suggested protocol for 10\u201315 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3\u02b9 adapter KXS013 was used for 3\u02b9 end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "tissue:embryo|treatment:N1", "GSM7716870", "GSM7716870: Fish embryo mRNA zfs:0000015 PAL seq v4; Danio rerio; OTHER", "GSM7716870 r1", "GSM7716870", "1", "Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37\u00b0C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3\u02b9 adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10\u201315 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3\u02b9 adapter KXS013 was used for 3\u02b9 end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "SRP455680", null, null, "Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep1_raw_read2.fastq.gz", "fastq fastq", 2695203962.0, 8779166.0, "GSM7716870 r1", "0:52 1:255", "A:684535386;C:669852151;G:719008886;T:614209811;N:7597728", 52, 255, null, null, 684535386, 669852151, 719008886, 614209811, 7597728, "SRX21396041", "SRS18636199", "SRA1694849", "Whitehead Institute", "Whitehead Institute", 2, 0.00117, 0.35837, 0.00017, 0.00682, 0.99859, 0.99963, 0.69473, 1.0, 52, 255, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "bulk", "bulk", null, "United States", "2023-08-17", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [25185, "SRR25670732", "SRX21396041", "SRS18636199", "SRP455680", "PRJNA1006406", "Control of polyA tail length and translation in vertebrate oocytes and early embryos", "GSE241107", "Other", "During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.", null, null, null, "Fish embryo mRNA zfs:0000015 PAL seq v4", "GSM7716870", null, "source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing", "Fish embryo mRNA zfs:0000015 PAL seq v4", "For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters \u201c m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7\u201d. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters \u201c  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate\u201d. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4\u20137 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4", "embryo", null, "Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37\u00b0C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3\u02b9 adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer\u2019s suggested protocol for 10\u201315 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3\u02b9 adapter KXS013 was used for 3\u02b9 end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "tissue:embryo|treatment:N1", "GSM7716870", "GSM7716870: Fish embryo mRNA zfs:0000015 PAL seq v4; Danio rerio; OTHER", "GSM7716870 r1", "GSM7716870", "1", "Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37\u00b0C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3\u02b9 adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10\u201315 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3\u02b9 adapter KXS013 was used for 3\u02b9 end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "SRP455680", null, null, "Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep2_raw_read2.fastq.gz Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep2_raw_read1.fastq.gz", "fastq fastq", 3476338446.0, 11323578.0, "GSM7716870 r2", "0:52 1:255", "A:841832234;C:898325199;G:968774318;T:760398070;N:7008625", 52, 255, null, null, 841832234, 898325199, 968774318, 760398070, 7008625, "SRX21396041", "SRS18636199", "SRA1694849", "Whitehead Institute", "Whitehead Institute", 2, 0.00245, 0.0, 0.00047, 0.0, 0.99803, 1.0, 0.58536, null, 52, 255, "T", "T", "mates < 9% mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "bulk", "bulk", "bulk", null, "United States", "2023-08-17", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"]], "truncated": false, "filtered_table_rows_count": 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