{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where experiment.library_layout = \"PAIRED\", technology = \"10x\" and tissue_curation = \"Spinal Cord\"", "rows": [[10176, "ERR5858457", "ERX5504346", "ERS6343450", "ERP128749", "PRJEB44676", "scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "E-MTAB-10390", "Transcriptome Analysis", "To analyse lesion induced gene regulation in progenitor cells at single cell resolution  we performed single cell RNAseq on FACS isolated her4.3:GFP progenitor cells from the spinal cord at 24 hours post lesion hpl post spinal injury at 3 dpf dpf  compared to age matched uninjured animals.", "ENA FIRST PUBLIC:2021 05 24|ENA LAST UPDATE:2021 05 24", null, "Protocols: Trunks containing the lesion sites or equivalent site from unlesioned fish are collected and kept in PBS on ice.Incubate the trunks up to 300 in 1 mL of 1X Trypsin EDTA at 37C for 5 7 mins.Stop dissociation by adding FBS to a final concentration of 5%.Centrifuge at 200g for 7 mins.Discard Sups.Resuspend in 500 uL of PBS.Add the cell suspension into a 40 uM cell strainer.Centrifuge at 200g for 7 mins.Resuspend in the buffer for FACS PBS 5% FBS Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines.", "Naive", "SAMEA8658904", "University Of Edinburgh", "ENA first public:2021 05 24|ENA last update:2021 05 24|External Id:SAMEA8658904|INSDC center alias:UOE|INSDC center name:University Of Edinburgh|INSDC first public:2021 05 24T00:14:32Z|INSDC last update:2021 05 24T00:14:32Z|INSDC status:public|Submitter Id:E MTAB 10390:Naive|age:4|broker name:ArrayExpress|cell type:ependymo radial glial cell|common name:zebrafish|developmental stage:larval day 4|immunophenotype:Her4.3+ positive|organism part:spinal cord|sample name:E MTAB 10390:Naive|sex:mixed|strain:WIK", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; scRNAseq of her4.3+ cells from lesi1d and unlesi1d zebrafish larvae spinal cord", "E MTAB 10390:Naive p", "Naive p", "scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "Trunks containing the lesion sites or equivalent site from unlesioned fish are collected and kept in PBS on ice.Incubate the trunks up to 300 in 1 mL of 1X Trypsin EDTA at 37C for 5 7 mins.Stop dissociation by adding FBS to a final concentration of 5%.Centrifuge at 200g for 7 mins.Discard Sups.Resuspend in 500 uL of PBS.Add the cell suspension into a 40 uM cell strainer.Centrifuge at 200g for 7 mins.Resuspend in the buffer for FACS PBS 5% FBS Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines  Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines.", "Experimental Factor: injury:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "Oligo-dT", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP128749", "Illumina NovaSeq 6000 paired end sequencing; scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "ENA FIRST PUBLIC:2021 05 24|ENA LAST UPDATE:2021 05 24", "Naive.bam", "bam", 44972162730.0, 499690697.0, "E MTAB 10390:Naive", "0:90", "A:13658826375;C:8733304343;G:9348137569;T:13228064343;N:3830100", 90, null, null, null, 13658826375, 8733304343, 9348137569, 13228064343, 3830100, "ERX5504346", "ERS6343450", "ERA4142789", "University Of Edinburgh|European Nucleotide Archive", "University Of Edinburgh|European Nucleotide Archive", 1, 0.89428, null, 0.32693, null, 0.75276, null, 0.5314, null, 90, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2021-05-24", "Larval", "Larval", "Spinal Cord", "Nervous System"], [10177, "ERR5858456", "ERX5504345", "ERS6343449", "ERP128749", "PRJEB44676", "scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "E-MTAB-10390", "Transcriptome Analysis", "To analyse lesion induced gene regulation in progenitor cells at single cell resolution  we performed single cell RNAseq on FACS isolated her4.3:GFP progenitor cells from the spinal cord at 24 hours post lesion hpl post spinal injury at 3 dpf dpf  compared to age matched uninjured animals.", "ENA FIRST PUBLIC:2021 05 24|ENA LAST UPDATE:2021 05 24", null, "Protocols: Trunks containing the lesion sites or equivalent site from unlesioned fish are collected and kept in PBS on ice.Incubate the trunks up to 300 in 1 mL of 1X Trypsin EDTA at 37C for 5 7 mins.Stop dissociation by adding FBS to a final concentration of 5%.Centrifuge at 200g for 7 mins.Discard Sups.Resuspend in 500 uL of PBS.Add the cell suspension into a 40 uM cell strainer.Centrifuge at 200g for 7 mins.Resuspend in the buffer for FACS PBS 5% FBS Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines.", "Lesi1d", "SAMEA8658903", "University Of Edinburgh", "ENA first public:2021 05 24|ENA last update:2021 05 24|External Id:SAMEA8658903|INSDC center alias:UOE|INSDC center name:University Of Edinburgh|INSDC first public:2021 05 24T00:14:32Z|INSDC last update:2021 05 24T00:14:32Z|INSDC status:public|Submitter Id:E MTAB 10390:Lesi1d|age:4|broker name:ArrayExpress|cell type:ependymo radial glial cell|common name:zebrafish|developmental stage:larval day 4|immunophenotype:Her4.3+ positive|injury:spinal injury lesion|organism part:spinal cord|sample name:E MTAB 10390:Lesi1d|sex:mixed|strain:WIK", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; scRNAseq of her4.3+ cells from lesi1d and unlesi1d zebrafish larvae spinal cord", "E MTAB 10390:Lesioned p", "Lesioned p", "scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "Trunks containing the lesion sites or equivalent site from unlesioned fish are collected and kept in PBS on ice.Incubate the trunks up to 300 in 1 mL of 1X Trypsin EDTA at 37C for 5 7 mins.Stop dissociation by adding FBS to a final concentration of 5%.Centrifuge at 200g for 7 mins.Discard Sups.Resuspend in 500 uL of PBS.Add the cell suspension into a 40 uM cell strainer.Centrifuge at 200g for 7 mins.Resuspend in the buffer for FACS PBS 5% FBS Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines  Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines.", "Experimental Factor: injury:spinal injury lesion", "RNA-Seq", "TRANSCRIPTOMIC", "Oligo-dT", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP128749", "Illumina NovaSeq 6000 paired end sequencing; scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "ENA FIRST PUBLIC:2021 05 24|ENA LAST UPDATE:2021 05 24", "Lesioned.bam", "bam", 49902588630.0, 554473207.0, "E MTAB 10390:Lesioned", "0:90", "A:14713351178;C:10191139050;G:10897887675;T:14095967201;N:4243526", 90, null, null, null, 14713351178, 10191139050, 10897887675, 14095967201, 4243526, "ERX5504345", "ERS6343449", "ERA4142789", "University Of Edinburgh|European Nucleotide Archive", "University Of Edinburgh|European Nucleotide Archive", 1, 0.91197, null, 0.29137, null, 0.7568, null, 0.56523, null, 90, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2021-05-24", "Larval", "Larval", "Spinal Cord", "Nervous System"], [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"], [60587, "SRR12424278", "SRX8920145", "SRS7176693", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 48hpf", "GSM4718658", null, "tissue:spinal cord cells|developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 48hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718658", "GSM4718658: scRNAseq olig2 eGFP 48hpf; Danio rerio; RNA Seq", "GSM4718658", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718658", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "olig2_egfp_48hpf_1_S41_L001_R1_001.fastq.gz olig2_egfp_48hpf_1_S41_L001_R2_001.fastq.gz", "fastq fastq", 18778651430.0, 62180965.0, "GSM4718658 r1", "0:151 1:151", "A:6542554044;C:3515052559;G:3489753648;T:5231107020;N:184159", 151, 151, null, null, 6542554044, 3515052559, 3489753648, 5231107020, 184159, "SRX8920145", "SRS7176693", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.90055, 0.0, 0.21162, 1.0, 0.77776, null, 0.53718, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [60588, "SRR12424279", "SRX8920145", "SRS7176693", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 48hpf", "GSM4718658", null, "tissue:spinal cord cells|developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 48hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718658", "GSM4718658: scRNAseq olig2 eGFP 48hpf; Danio rerio; RNA Seq", "GSM4718658", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718658", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "olig2_egfp_48hpf_2_S42_L001_R1_001.fastq.gz olig2_egfp_48hpf_2_S42_L001_R2_001.fastq.gz", "fastq fastq", 22242782898.0, 73651599.0, "GSM4718658 r2", "0:151 1:151", "A:7753674261;C:4160150132;G:4127581173;T:6201157562;N:219770", 151, 151, null, null, 7753674261, 4160150132, 4127581173, 6201157562, 219770, "SRX8920145", "SRS7176693", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.90097, 0.0, 0.2127, 1.0, 0.77966, null, 0.54192, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [60589, "SRR12424280", "SRX8920145", "SRS7176693", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 48hpf", "GSM4718658", null, "tissue:spinal cord cells|developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 48hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718658", "GSM4718658: scRNAseq olig2 eGFP 48hpf; Danio rerio; RNA Seq", "GSM4718658", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718658", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "olig2_egfp_48hpf_3_S43_L001_R2_001.fastq.gz olig2_egfp_48hpf_3_S43_L001_R1_001.fastq.gz", "fastq fastq", 25591190986.0, 84739043.0, "GSM4718658 r3", "0:151 1:151", "A:8917343424;C:4797392797;G:4757647263;T:7118554564;N:252938", 151, 151, null, null, 8917343424, 4797392797, 4757647263, 7118554564, 252938, "SRX8920145", "SRS7176693", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.90089, 0.0, 0.2095, 1.0, 0.78078, null, 0.53755, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [60590, "SRR12424281", "SRX8920145", "SRS7176693", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 48hpf", "GSM4718658", null, "tissue:spinal cord cells|developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 48hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718658", "GSM4718658: scRNAseq olig2 eGFP 48hpf; Danio rerio; RNA Seq", "GSM4718658", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718658", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "olig2_egfp_48hpf_4_S44_L001_R1_001.fastq.gz olig2_egfp_48hpf_4_S44_L001_R2_001.fastq.gz", "fastq fastq", 25546281170.0, 84590335.0, "GSM4718658 r4", "0:151 1:151", "A:8905009196;C:4774524700;G:4734395060;T:7132103501;N:248713", 151, 151, null, null, 8905009196, 4774524700, 4734395060, 7132103501, 248713, "SRX8920145", "SRS7176693", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.90169, 0.0, 0.21158, 1.0, 0.77697, null, 0.54006, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [60591, "SRR12424274", "SRX8920144", "SRS7176692", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 36hpf", "GSM4718657", null, "tissue:spinal cord cells|developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 36hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718657", "GSM4718657: scRNAseq olig2 eGFP 36hpf; Danio rerio; RNA Seq", "GSM4718657", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718657", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "Olig2_eGFP_36hfp_1_S69_L001_R1_001.fastq.gz Olig2_eGFP_36hfp_1_S69_L001_R2_001.fastq.gz", "fastq fastq", 13366864582.0, 44261141.0, "GSM4718657 r1", "0:151 1:151", "A:3721647646;C:2385939960;G:3494552257;T:3764514262;N:210457", 151, 151, null, null, 3721647646, 2385939960, 3494552257, 3764514262, 210457, "SRX8920144", "SRS7176692", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.85449, 0.0, 0.08264, 1.0, 0.84086, null, 0.50156, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [60592, "SRR12424275", "SRX8920144", "SRS7176692", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 36hpf", "GSM4718657", null, "tissue:spinal cord cells|developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 36hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718657", "GSM4718657: scRNAseq olig2 eGFP 36hpf; Danio rerio; RNA Seq", "GSM4718657", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718657", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "Olig2_eGFP_36hfp_2_S70_L001_R1_001.fastq.gz Olig2_eGFP_36hfp_2_S70_L001_R2_001.fastq.gz", "fastq fastq", 10606211914.0, 35119907.0, "GSM4718657 r2", "0:151 1:151", "A:2974656607;C:1869869441;G:2750621038;T:3010898673;N:166155", 151, 151, null, null, 2974656607, 1869869441, 2750621038, 3010898673, 166155, "SRX8920144", "SRS7176692", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.84822, 0.0, 0.08456, 1.0, 0.83719, null, 0.51503, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [60593, "SRR12424276", "SRX8920144", "SRS7176692", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 36hpf", "GSM4718657", null, "tissue:spinal cord cells|developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 36hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718657", "GSM4718657: scRNAseq olig2 eGFP 36hpf; Danio rerio; RNA Seq", "GSM4718657", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718657", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "Olig2_eGFP_36hfp_3_S71_L001_R1_001.fastq.gz Olig2_eGFP_36hfp_3_S71_L001_R2_001.fastq.gz", "fastq fastq", 15614508642.0, 51703671.0, "GSM4718657 r3", "0:151 1:151", "A:4330312984;C:2826855730;G:4093245712;T:4363847925;N:246291", 151, 151, null, null, 4330312984, 2826855730, 4093245712, 4363847925, 246291, "SRX8920144", "SRS7176692", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.8543, 0.0, 0.08219, 1.0, 0.83727, null, 0.51892, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [60594, "SRR12424277", "SRX8920144", "SRS7176692", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 36hpf", "GSM4718657", null, "tissue:spinal cord cells|developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 36hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718657", "GSM4718657: scRNAseq olig2 eGFP 36hpf; Danio rerio; RNA Seq", "GSM4718657", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718657", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "Olig2_eGFP_36hfp_4_S72_L001_R1_001.fastq.gz Olig2_eGFP_36hfp_4_S72_L001_R2_001.fastq.gz", "fastq fastq", 10707787198.0, 35456249.0, "GSM4718657 r4", "0:151 1:151", "A:2981466900;C:1909696186;G:2797297473;T:3019156550;N:170089", 151, 151, null, null, 2981466900, 1909696186, 2797297473, 3019156550, 170089, "SRX8920144", "SRS7176692", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.85466, 0.0, 0.08381, 1.0, 0.83562, null, 0.51035, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [60595, "SRR12424270", "SRX8920143", "SRS7176691", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 24hpf", "GSM4718656", null, "tissue:spinal cord cells|developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 24hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718656", "GSM4718656: scRNAseq olig2 eGFP 24hpf; Danio rerio; RNA Seq", "GSM4718656", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718656", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "Olig2_eGFP_24hfp_1_S10_L001_R1_001.fastq.gz Olig2_eGFP_24hfp_1_S10_L001_R2_001.fastq.gz", "fastq fastq", 21279021170.0, 70460335.0, "GSM4718656 r1", "0:151 1:151", "A:5842145219;C:3712477572;G:5933108587;T:5790922441;N:367351", 151, 151, null, null, 5842145219, 3712477572, 5933108587, 5790922441, 367351, "SRX8920143", "SRS7176691", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.83071, 0.0, 0.06828, 1.0, 0.8406, null, 0.5078, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [60596, "SRR12424271", "SRX8920143", "SRS7176691", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 24hpf", "GSM4718656", null, "tissue:spinal cord cells|developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 24hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718656", "GSM4718656: scRNAseq olig2 eGFP 24hpf; Danio rerio; RNA Seq", "GSM4718656", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718656", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "Olig2_eGFP_24hfp_2_S20_L001_R1_001.fastq.gz Olig2_eGFP_24hfp_2_S20_L001_R2_001.fastq.gz", "fastq fastq", 22414094210.0, 74218855.0, "GSM4718656 r2", "0:151 1:151", "A:6145677154;C:3904876630;G:6254105391;T:6109049174;N:385861", 151, 151, null, null, 6145677154, 3904876630, 6254105391, 6109049174, 385861, "SRX8920143", "SRS7176691", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.83148, 0.0, 0.06914, 1.0, 0.84033, null, 0.49666, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [60597, "SRR12424272", "SRX8920143", "SRS7176691", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 24hpf", "GSM4718656", null, "tissue:spinal cord cells|developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 24hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718656", "GSM4718656: scRNAseq olig2 eGFP 24hpf; Danio rerio; RNA Seq", "GSM4718656", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718656", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "Olig2_eGFP_24hfp_3_S30_L001_R1_001.fastq.gz Olig2_eGFP_24hfp_3_S30_L001_R2_001.fastq.gz", "fastq fastq", 16406166006.0, 54325053.0, "GSM4718656 r3", "0:151 1:151", "A:4513103872;C:2865727260;G:4569430659;T:4457621166;N:283049", 151, 151, null, null, 4513103872, 2865727260, 4569430659, 4457621166, 283049, "SRX8920143", "SRS7176691", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.82928, 0.0, 0.06878, 1.0, 0.83989, null, 0.4916, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [60598, "SRR12424273", "SRX8920143", "SRS7176691", "SRP276929", "PRJNA656271", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish", "GSE155988", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:32680935", null, "scRNAseq olig2 eGFP 24hpf", "GSM4718656", null, "tissue:spinal cord cells|developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 24hpf", "Raw sequencing reads were demultiplexed  mapped to the zebrafish reference genome and summarized into gene expression matrices using CellRanger version 3.0.1 Count matrices were further filtered in Seurat 3.1.0 https://satijalab.org/seurat/ to remove cell barcodes with fewer than 250 detectable genes  more than 5% of UMIs derived from mitochondrial genes  or more than 50 000 UMIs to exclude putative doublets Standard Seurat normalization and PCA was run using the 1 291 most variable genes Applied Harmony https://doi.org/10.1038/s41592 019 0619 0 alignment theta = 2 to correct for inter sample variation  chose 20 Harmony dimensions and applied Seurat UMAP reduction Genome build: GRCz11 Supplementary files format and content: gzipped csv file of matrix of normalized gene expression by cell for merged 3 scRNAseq samples Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "GSM4718656", "GSM4718656: scRNAseq olig2 eGFP 24hpf; Danio rerio; RNA Seq", "GSM4718656", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM4718656", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP276929", null, null, "Olig2_eGFP_24hfp_4_S40_L001_R1_001.fastq.gz Olig2_eGFP_24hfp_4_S40_L001_R2_001.fastq.gz", "fastq fastq", 17382967792.0, 57559496.0, "GSM4718656 r4", "0:151 1:151", "A:4767137063;C:3044788720;G:4846983492;T:4723757048;N:301469", 151, 151, null, null, 4767137063, 3044788720, 4846983492, 4723757048, 301469, "SRX8920143", "SRS7176691", "SRA1111061", "GEO", "University of Colorado School of Medicine", 2, 0.0, 0.83084, 0.0, 0.06779, 1.0, 0.83948, null, 0.49689, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2020-08-10", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [62942, "SRR13447021", "SRX9860257", "SRS8040688", "SRP301983", "PRJNA692494", "Localized EMT reprograms glial progenitors to promote spinal cord repair [single cell RNA seq]", "GSE164944", "Other", "Single nuclear RNA sequencing was performed on spinal cord tissues from Tggfap:EGFP zebrafish at 1 xxx post injury using the 10x Genomics platform. Overall design: Single nuclear RNA sequencing of spinal cord tissue.", "parent bioproject:PRJNA692492", "pubmed:33609461", null, "single cell injured spinal cord", "GSM5023610", null, "tissue:5 xxx post injury spinal cord nuclear|tissues:spinal cord|rna population:nuclear|time point:5 xxx post injury", "single cell injured spinal cord", "Aligned reads were analyzed using the Seurat package v3.1.4 within R v3.6.3. Cells that have unique number of genes between 200 to 4000 and a mitochondrial gene percentage less than five were filtered. The \u201cSCTransform\u201d function was used for normalization  scaling and finding variable features. SCTransform returned 3000 highly variable features for downstream analysis. Significant principal components were determined using \u201cElbowPlot\u201d function. Forty principal components were selected to create a Shared Nearest Neighbour SNN graph using the \u201cFindNeighbours\u201d function. Clustering was performed by the Louvain algorithm using the function \u201cFindClusters\u201d and the resolution parameter was set to one. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 40 principle components and to visualize the data in two dimensional space \u201cRunUMAP\u201d function Differential gene expression for individual cluster was identified using Wilcoxon rank sum tests in the \u201cFindAllMarkers\u201d function. Supplementary files format and content: RDS file", "5 xxx post injury spinal cord nuclear", "Adult zebrafish were subjected to complete spinal cord transection.", "gfap:EGFP transgenic animals were subjected to SCI and 2 mm SC tissue sections spanning the lesion site were collected at 1 wpi. Nuclei were isolated from 45 pooled SC tissues. For single nuclear library preparation on the 10x Genomics platform  the Chromium Single Cell 3\u2032 GEM Library and Gel Bead Kit v3 cat# 1000092 and the Chromium Chip B Single Cell Kit cat# 1000074 were used according to the manufacturer\u2019s instructions in the Chromium Single Cell 3\u2032 Reagents Kits V3 User Guide. The resulting cDNA libraries were quantified on an Agilent Tapestation and sequenced on Illumina HiSeq 3000.", null, "tissues:spinal cord|rna population:nuclear|time point:5 xxx post injury", "GSM5023610", "GSM5023610: single cell injured spinal cord; Danio rerio; RNA Seq", "GSM5023610", null, "1", "gfap:EGFP transgenic animals were subjected to SCI and 2 mm SC tissue sections spanning the lesion site were collected at 1 wpi. Nuclei were isolated from 45 pooled SC tissues. For single nuclear library preparation on the 10x Genomics platform  the Chromium Single Cell 3\u2032 GEM Library and Gel Bead Kit v3 cat# 1000092 and the Chromium Chip B Single Cell Kit cat# 1000074 were used according to the manufacturer's instructions in the Chromium Single Cell 3\u2032 Reagents Kits V3 User Guide. The resulting cDNA libraries were quantified on an Agilent Tapestation and sequenced on Illumina HiSeq 3000.", "GEO Accession:GSM5023610", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 3000", null, "SRP301983", null, "loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=ZB nuc S1 L007 I1 001.fastq.gz   read2PairFiles=ZB nuc S1 L007 R1 001.fastq.gz   read3PairFiles=ZB nuc S1 L007 R2 001.fastq.gz", "ZB_nuc_S1_L007_R2_001.fastq.gz ZB_nuc_S1_L007_R1_001.fastq.gz ZB_nuc_S1_L007_I1_001.fastq.gz", "fastq fastq fastq", 134333186988.0, 436146711.0, "GSM5023610 r1", "0:8 1:150 2:150", "A:34573306207;C:23174725516;G:24790836286;T:51771968988;N:22349991", 8, 150, 150, null, 34573306207, 23174725516, 24790836286, 51771968988, 22349991, "SRX9860257", "SRS8040688", "SRA1184553", "GEO", "Developmental Biology, Washington University", 1, 0.72186, null, 0.41754, null, 0.80551, null, 0.54979, null, 150, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-01-15", "Adult", "Adult", "Spinal Cord", "Nervous System"], [64205, "SRR14326888", "SRX10681834", "SRS8775213", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 48hpf [reanalysis]", "GSM5266260", null, "tissue:spinal cord cells|developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 48hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266260", "GSM5266260: scRNAseq olig2 eGFP 48hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266260", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266260", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "olig2_egfp_48hpf_1_S41_L001_R1_001.fastq olig2_egfp_48hpf_1_S41_L001_R2_001.fastq", "fastq fastq", 18778651430.0, 62180965.0, "GSM5266260 r1", "0:151 1:151", "A:6542554044;C:3515052559;G:3489753648;T:5231107020;N:184159", 151, 151, null, null, 6542554044, 3515052559, 3489753648, 5231107020, 184159, "SRX10681834", "SRS8775213", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.90056, 0.0, 0.21167, 1.0, 0.7781, null, 0.53738, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [64206, "SRR14326889", "SRX10681834", "SRS8775213", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 48hpf [reanalysis]", "GSM5266260", null, "tissue:spinal cord cells|developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 48hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266260", "GSM5266260: scRNAseq olig2 eGFP 48hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266260", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266260", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "olig2_egfp_48hpf_2_S42_L001_R1_001.fastq olig2_egfp_48hpf_2_S42_L001_R2_001.fastq", "fastq fastq", 22242782898.0, 73651599.0, "GSM5266260 r2", "0:151 1:151", "A:7753674261;C:4160150132;G:4127581173;T:6201157562;N:219770", 151, 151, null, null, 7753674261, 4160150132, 4127581173, 6201157562, 219770, "SRX10681834", "SRS8775213", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.90096, 0.0, 0.21256, 1.0, 0.77928, null, 0.54059, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [64207, "SRR14326890", "SRX10681834", "SRS8775213", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 48hpf [reanalysis]", "GSM5266260", null, "tissue:spinal cord cells|developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 48hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266260", "GSM5266260: scRNAseq olig2 eGFP 48hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266260", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266260", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "olig2_egfp_48hpf_3_S43_L001_R2_001.fastq olig2_egfp_48hpf_3_S43_L001_R1_001.fastq", "fastq fastq", 25591190986.0, 84739043.0, "GSM5266260 r3", "0:151 1:151", "A:8917343424;C:4797392797;G:4757647263;T:7118554564;N:252938", 151, 151, null, null, 8917343424, 4797392797, 4757647263, 7118554564, 252938, "SRX10681834", "SRS8775213", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.90089, 0.0, 0.20934, 1.0, 0.78086, null, 0.53725, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [64208, "SRR14326891", "SRX10681834", "SRS8775213", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 48hpf [reanalysis]", "GSM5266260", null, "tissue:spinal cord cells|developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 48hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:48 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266260", "GSM5266260: scRNAseq olig2 eGFP 48hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266260", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266260", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "olig2_egfp_48hpf_4_S44_L001_R1_001.fastq olig2_egfp_48hpf_4_S44_L001_R2_001.fastq", "fastq fastq", 25546281170.0, 84590335.0, "GSM5266260 r4", "0:151 1:151", "A:8905009196;C:4774524700;G:4734395060;T:7132103501;N:248713", 151, 151, null, null, 8905009196, 4774524700, 4734395060, 7132103501, 248713, "SRX10681834", "SRS8775213", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.90171, 0.0, 0.21154, 1.0, 0.77674, null, 0.53902, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [64209, "SRR14326884", "SRX10681833", "SRS8775212", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 36hpf [reanalysis]", "GSM5266259", null, "tissue:spinal cord cells|developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 36hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266259", "GSM5266259: scRNAseq olig2 eGFP 36hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266259", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266259", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "Olig2_eGFP_36hfp_1_S69_L001_R1_001.fastq Olig2_eGFP_36hfp_1_S69_L001_R2_001.fastq", "fastq fastq", 13366864582.0, 44261141.0, "GSM5266259 r1", "0:151 1:151", "A:3721647646;C:2385939960;G:3494552257;T:3764514262;N:210457", 151, 151, null, null, 3721647646, 2385939960, 3494552257, 3764514262, 210457, "SRX10681833", "SRS8775212", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.85451, 0.0, 0.08269, 1.0, 0.84094, null, 0.5016, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [64210, "SRR14326885", "SRX10681833", "SRS8775212", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 36hpf [reanalysis]", "GSM5266259", null, "tissue:spinal cord cells|developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 36hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266259", "GSM5266259: scRNAseq olig2 eGFP 36hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266259", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266259", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "Olig2_eGFP_36hfp_2_S70_L001_R1_001.fastq Olig2_eGFP_36hfp_2_S70_L001_R2_001.fastq", "fastq fastq", 10606211914.0, 35119907.0, "GSM5266259 r2", "0:151 1:151", "A:2974656607;C:1869869441;G:2750621038;T:3010898673;N:166155", 151, 151, null, null, 2974656607, 1869869441, 2750621038, 3010898673, 166155, "SRX10681833", "SRS8775212", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.84821, 0.0, 0.08459, 1.0, 0.83731, null, 0.51513, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [64211, "SRR14326886", "SRX10681833", "SRS8775212", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 36hpf [reanalysis]", "GSM5266259", null, "tissue:spinal cord cells|developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 36hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266259", "GSM5266259: scRNAseq olig2 eGFP 36hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266259", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266259", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "Olig2_eGFP_36hfp_3_S71_L001_R1_001.fastq Olig2_eGFP_36hfp_3_S71_L001_R2_001.fastq", "fastq fastq", 15614508642.0, 51703671.0, "GSM5266259 r3", "0:151 1:151", "A:4330312984;C:2826855730;G:4093245712;T:4363847925;N:246291", 151, 151, null, null, 4330312984, 2826855730, 4093245712, 4363847925, 246291, "SRX10681833", "SRS8775212", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.85427, 0.0, 0.08218, 1.0, 0.83713, null, 0.51889, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [64212, "SRR14326887", "SRX10681833", "SRS8775212", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 36hpf [reanalysis]", "GSM5266259", null, "tissue:spinal cord cells|developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 36hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:36 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266259", "GSM5266259: scRNAseq olig2 eGFP 36hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266259", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266259", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "Olig2_eGFP_36hfp_4_S72_L001_R1_001.fastq Olig2_eGFP_36hfp_4_S72_L001_R2_001.fastq", "fastq fastq", 10707787198.0, 35456249.0, "GSM5266259 r4", "0:151 1:151", "A:2981466900;C:1909696186;G:2797297473;T:3019156550;N:170089", 151, 151, null, null, 2981466900, 1909696186, 2797297473, 3019156550, 170089, "SRX10681833", "SRS8775212", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.85465, 0.0, 0.08381, 1.0, 0.8355, null, 0.51049, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [64213, "SRR14326880", "SRX10681832", "SRS8775211", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 24hpf [reanalysis]", "GSM5266258", null, "tissue:spinal cord cells|developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 24hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266258", "GSM5266258: scRNAseq olig2 eGFP 24hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266258", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266258", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "Olig2_eGFP_24hfp_1_S10_L001_R1_001.fastq Olig2_eGFP_24hfp_1_S10_L001_R2_001.fastq", "fastq fastq", 21279021170.0, 70460335.0, "GSM5266258 r1", "0:151 1:151", "A:5842145219;C:3712477572;G:5933108587;T:5790922441;N:367351", 151, 151, null, null, 5842145219, 3712477572, 5933108587, 5790922441, 367351, "SRX10681832", "SRS8775211", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.83071, 0.0, 0.06834, 1.0, 0.8407, null, 0.50743, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [64214, "SRR14326881", "SRX10681832", "SRS8775211", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 24hpf [reanalysis]", "GSM5266258", null, "tissue:spinal cord cells|developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 24hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266258", "GSM5266258: scRNAseq olig2 eGFP 24hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266258", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266258", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "Olig2_eGFP_24hfp_2_S20_L001_R1_001.fastq Olig2_eGFP_24hfp_2_S20_L001_R2_001.fastq", "fastq fastq", 22414094210.0, 74218855.0, "GSM5266258 r2", "0:151 1:151", "A:6145677154;C:3904876630;G:6254105391;T:6109049174;N:385861", 151, 151, null, null, 6145677154, 3904876630, 6254105391, 6109049174, 385861, "SRX10681832", "SRS8775211", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.83142, 0.0, 0.06899, 1.0, 0.84029, null, 0.49666, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [64215, "SRR14326882", "SRX10681832", "SRS8775211", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 24hpf [reanalysis]", "GSM5266258", null, "tissue:spinal cord cells|developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 24hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266258", "GSM5266258: scRNAseq olig2 eGFP 24hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266258", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266258", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "Olig2_eGFP_24hfp_3_S30_L001_R1_001.fastq Olig2_eGFP_24hfp_3_S30_L001_R2_001.fastq", "fastq fastq", 16406166006.0, 54325053.0, "GSM5266258 r3", "0:151 1:151", "A:4513103872;C:2865727260;G:4569430659;T:4457621166;N:283049", 151, 151, null, null, 4513103872, 2865727260, 4569430659, 4457621166, 283049, "SRX10681832", "SRS8775211", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.8293, 0.0, 0.06878, 1.0, 0.84017, null, 0.49152, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [64216, "SRR14326883", "SRX10681832", "SRS8775211", "SRP316409", "PRJNA725224", "Single cell RNA seq analysis of pMN neural progenitors from zebrafish [Reanalysis of GSE155988 data set]", "GSE173350", "Transcriptome Analysis", "The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so  we sorted fluorescently marked pMN cells obtained from Tgolig2:EGFP zebrafish embryos at 24  36 hpf and 48 hpf and performed 10X Chromium single cell RNA seq. Overall design: Three samples obtained from wild type zebrafish embryos at three developmental timepoints were analyzed.", null, "pubmed:34303700", null, "scRNAseq olig2 eGFP 24hpf [reanalysis]", "GSM5266258", null, "tissue:spinal cord cells|developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "scRNAseq olig2 eGFP 24hpf [reanalysis]", "Generated cell feature counts for each single cell RNAseq library using cellranger count   transcriptome=GRCz11 Each single cell RNAseq sample was converted to a Seurat object using the Seurat Read 10X  CreateSeuratObject functions 3 separate Seurat objects filtered individual Seurat objects to select cells with nGene >250 and percent.mitochondrial <5% and nFeature RNA <50000 hpf 24 hpf and 48 hpf samples or <30000 hpf 36 hpf Processed and filtered Seurat objects using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction   'pca'  dims = 1:40  FindClustersresolution= 0.9 hpf 48 hpf; 1 hpf 36 hpf; 1.2 hpf 24 hpf  dims = 1:40 and RunUMAPdims=1:40 Clusters with low nFeatures and high percent.mitochondrial reads were removed as dying cells in 24 hpf and 36 hpf datasets Individual processed Seurat objects from each sample were intergrated using the FindIntergrationAnchorsdims 1:40 and IntergrateDatadims = 1:40 Seurat functions Processed and filtered intergrated Seurat object using Seurat NormalizeData  FindVariableFeaturesselection.method = 'vst'  nfeatures = 2000  ScaleData vars.to.regress=c'nCount RNA' 'percent.mito' 'CC.difference'  RunPCAnpcs=100  RunUMAPdims = 1:40  FindNeighborsreduction = 'pca'  dims = 1:40  FindClustersresolution=  1.2  dims = 1:40 and RunUMAPdims=1:40 Genome build: GRCz11 Supplementary files format and content: gzipped rds file of intergrated Seurat object Supplementary files format and content: gzipped csv file of metadata for cells", "spinal cord cells", "No treatment was applied to the samples.", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.\u00a0 Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.\u00a0", "Embryos were raised at 28.5\u00b0C in E3 media 5 mM NaCl  0.17 mM KCl  0.33 mM CaCl2  0.33 mM MgSO4 at pH 7.4  with sodium bicarbonate  sorted for good health and staged accordingly to developmental morphological features and hpf", "developmental stage:24 hpf ZFIN ID: ZDB ALT 041129 8", "GSM5266258", "GSM5266258: scRNAseq olig2 eGFP 24hpf [reanalysis]; Danio rerio; RNA Seq", "GSM5266258", null, "1", "24  36  and 48 hpf Tgolig2:EGFP euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 \u03bcl of pre chilled Ca free Ringers solution 116 mM NaCl  2.6 mM KCl  5 mM HEPES  pH 7.0 on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 \u03bcl of protease solution 10 mg/ml BI protease  125 U/ml DNase  2.5 mM EDTA  1X PBS was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 \u03bcl of STOP solution 30% FBS  0.8 mM CaCl2  1X PBS was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4\u00b0C and supernatant was removed. On ice  1 ml of chilled suspension media 1% FBS  0.8 mM CaCl2  50 U/ml Penicillin  0.05 mg/ml Streptomycin was added to samples and then spun down again at 400g for 5 min at 4\u00b0C. Supernatant was removed  and 400 \u03bcl of chilled suspension media was added and solution was filtered through a 35 \u03bcm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 \u03bcl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell three prime Reagent Kit part no. PN 1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument.", "GEO Accession:GSM5266258", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP316409", null, null, "Olig2_eGFP_24hfp_4_S40_L001_R1_001.fastq Olig2_eGFP_24hfp_4_S40_L001_R2_001.fastq", "fastq fastq", 17382967792.0, 57559496.0, "GSM5266258 r4", "0:151 1:151", "A:4767137063;C:3044788720;G:4846983492;T:4723757048;N:301469", 151, 151, null, null, 4767137063, 3044788720, 4846983492, 4723757048, 301469, "SRX10681832", "SRS8775211", "SRA1223938", "GEO", "Bruce Appel, Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus", 2, 0.0, 0.83086, 0.0, 0.0679, 1.0, 0.83944, null, 0.4968, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-04-26", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [71409, "SRR21590947", "SRX17592668", "SRS15130333", "SRP397443", "PRJNA880627", "Single cell sequencing of WT and hb egfaKO zebrafish spinal cord  at 1 week post sham or transection injury.", "GSE213435", "Transcriptome Analysis", "We identified hb egfa as a secreted factor necessary and sufficient for spinal cord regeneration in zebrafish. To elucidate the Hb egfa mechanism of action  we perforemd single cell sequencing of WT and hb egfaKO zebrafish spinal cord. Overall design: Adult zebrafish were subjected to spinal cord transection. 2 mm rostral and caudal to the transection site were collected at 1 xxx post injury. Spinal cord of sham injured animals was collected as control. Samples were and subjected to the 10X Genomics pipeline for single cell sequencing 4 samples in total  WT 1 wpi  WT sham  hb egfaKO 1 wpi  hb egfaKO sham.", null, "pubmed:37567873", null, "danRer11 WT 7dpi", "GSM6586115", null, "source name:spinal cord|genotype:WT|tissue:spinal cord|strain:Ekkwill EK|age:6 month|treatment:7dpi|geo loc name:missing|collection date:missing", "danRer11 WT 7dpi", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the Cell Ranger software v6.0.1 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/what is cell ranger Assembly: danRer11 Supplementary files format and content: h5 files for count table", "spinal cord", null, "Samples were processed following the 10X Chromium platform following the manufacturer\u2019s guidelines  using 10X Single Cell 3\u2019 v3 chemistry 10X Genomics  Pleasanton  USA.", null, "genotype:WT|tissue:spinal cord|strain:Ekkwill EK|age:6 month|treatment:7dpi", "GSM6586115", "GSM6586115: danRer11 WT 7dpi; Danio rerio; RNA Seq", "GSM6586115 r1", "GSM6586115", "1", "Samples were processed following the 10X Chromium platform following the manufacturer's guidelines  using 10X Single Cell three prime v3 chemistry 10X Genomics  Pleasanton  USA.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "DNBSEQ", "DNBSEQ-G400", null, "SRP397443", null, null, "KP_VC_sample4_S1_L001_R2_001.fastq.gz KP_VC_sample4_S1_L001_R1_001.fastq.gz", "fastq fastq", 50454681480.0, 423988920.0, "GSM6586115 r1", "0:28 1:91", "A:14159328348;C:10820572193;G:11486998159;T:13985883249;N:1899531", 28, 91, null, null, 14159328348, 10820572193, 11486998159, 13985883249, 1899531, "SRX17592668", "SRS15130333", "SRA1500311", "Cell biology, Duke University", "Poss, Cell Biology, Duke University", 2, 0.00753, 0.88935, 0.00224, 0.18283, 0.98528, 0.77656, 0.24704, 0.5609, 28, 91, "T", "B", "sc-like readlen", "bgi", "bgi", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2022-09-15", "Adult", "Adult", "Spinal Cord", "Nervous System"], [71410, "SRR21590948", "SRX17592667", "SRS15130332", "SRP397443", "PRJNA880627", "Single cell sequencing of WT and hb egfaKO zebrafish spinal cord  at 1 week post sham or transection injury.", "GSE213435", "Transcriptome Analysis", "We identified hb egfa as a secreted factor necessary and sufficient for spinal cord regeneration in zebrafish. To elucidate the Hb egfa mechanism of action  we perforemd single cell sequencing of WT and hb egfaKO zebrafish spinal cord. Overall design: Adult zebrafish were subjected to spinal cord transection. 2 mm rostral and caudal to the transection site were collected at 1 xxx post injury. Spinal cord of sham injured animals was collected as control. Samples were and subjected to the 10X Genomics pipeline for single cell sequencing 4 samples in total  WT 1 wpi  WT sham  hb egfaKO 1 wpi  hb egfaKO sham.", null, "pubmed:37567873", null, "danRer11 WT sham", "GSM6586114", null, "source name:spinal cord|genotype:WT|tissue:spinal cord|strain:Ekkwill EK|age:6 month|treatment:sham|geo loc name:missing|collection date:missing", "danRer11 WT sham", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the Cell Ranger software v6.0.1 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/what is cell ranger Assembly: danRer11 Supplementary files format and content: h5 files for count table", "spinal cord", null, "Samples were processed following the 10X Chromium platform following the manufacturer\u2019s guidelines  using 10X Single Cell 3\u2019 v3 chemistry 10X Genomics  Pleasanton  USA.", null, "genotype:WT|tissue:spinal cord|strain:Ekkwill EK|age:6 month|treatment:sham", "GSM6586114", "GSM6586114: danRer11 WT sham; Danio rerio; RNA Seq", "GSM6586114 r1", "GSM6586114", "1", "Samples were processed following the 10X Chromium platform following the manufacturer's guidelines  using 10X Single Cell three prime v3 chemistry 10X Genomics  Pleasanton  USA.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "DNBSEQ", "DNBSEQ-G400", null, "SRP397443", null, null, "KP_VC_sample3_S1_L001_R1_001.fastq.gz KP_VC_sample3_S1_L001_R2_001.fastq.gz", "fastq fastq", 49434606578.0, 415416862.0, "GSM6586114 r1", "0:28 1:91", "A:13689985840;C:10750469002;G:11165716773;T:13825714788;N:2720175", 28, 91, null, null, 13689985840, 10750469002, 11165716773, 13825714788, 2720175, "SRX17592667", "SRS15130332", "SRA1500311", "Cell biology, Duke University", "Poss, Cell Biology, Duke University", 2, 0.01019, 0.90181, 0.00309, 0.16838, 0.98522, 0.78459, 0.27523, 0.51926, 28, 91, "T", "B", "sc-like readlen", "bgi", "bgi", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2022-09-15", "Adult", "Adult", "Spinal Cord", "Nervous System"], [71411, "SRR21590949", "SRX17592666", "SRS15130331", "SRP397443", "PRJNA880627", "Single cell sequencing of WT and hb egfaKO zebrafish spinal cord  at 1 week post sham or transection injury.", "GSE213435", "Transcriptome Analysis", "We identified hb egfa as a secreted factor necessary and sufficient for spinal cord regeneration in zebrafish. To elucidate the Hb egfa mechanism of action  we perforemd single cell sequencing of WT and hb egfaKO zebrafish spinal cord. Overall design: Adult zebrafish were subjected to spinal cord transection. 2 mm rostral and caudal to the transection site were collected at 1 xxx post injury. Spinal cord of sham injured animals was collected as control. Samples were and subjected to the 10X Genomics pipeline for single cell sequencing 4 samples in total  WT 1 wpi  WT sham  hb egfaKO 1 wpi  hb egfaKO sham.", null, "pubmed:37567873", null, "danRer11 hbegfaKO 7dpi", "GSM6586113", null, "source name:spinal cord|genotype:hbegfaKO|tissue:spinal cord|strain:Ekkwill EK|age:6 month|treatment:7dpi|geo loc name:missing|collection date:missing", "danRer11 hbegfaKO 7dpi", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the Cell Ranger software v6.0.1 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/what is cell ranger Assembly: danRer11 Supplementary files format and content: h5 files for count table", "spinal cord", null, "Samples were processed following the 10X Chromium platform following the manufacturer\u2019s guidelines  using 10X Single Cell 3\u2019 v3 chemistry 10X Genomics  Pleasanton  USA.", null, "genotype:hbegfaKO|tissue:spinal cord|strain:Ekkwill EK|age:6 month|treatment:7dpi", "GSM6586113", "GSM6586113: danRer11 hbegfaKO 7dpi; Danio rerio; RNA Seq", "GSM6586113 r1", "GSM6586113", "1", "Samples were processed following the 10X Chromium platform following the manufacturer's guidelines  using 10X Single Cell three prime v3 chemistry 10X Genomics  Pleasanton  USA.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "DNBSEQ", "DNBSEQ-G400", null, "SRP397443", null, null, "KP_VC_sample2_S1_L001_R1_001.fastq.gz KP_VC_sample2_S1_L001_R2_001.fastq.gz", "fastq fastq", 48346471411.0, 406272869.0, "GSM6586113 r1", "0:28 1:91", "A:13278513523;C:10826562396;G:11318717473;T:12918171753;N:4506266", 28, 91, null, null, 13278513523, 10826562396, 11318717473, 12918171753, 4506266, "SRX17592666", "SRS15130331", "SRA1500311", "Cell biology, Duke University", "Poss, Cell Biology, Duke University", 2, 0.01379, 0.89003, 0.00446, 0.1909, 0.97808, 0.78415, 0.32888, 0.6007, 28, 91, "T", "B", "sc-like readlen", "bgi", "bgi", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2022-09-15", "Adult", "Adult", "Spinal Cord", "Nervous System"], [71412, "SRR21590950", "SRX17592665", "SRS15130330", "SRP397443", "PRJNA880627", "Single cell sequencing of WT and hb egfaKO zebrafish spinal cord  at 1 week post sham or transection injury.", "GSE213435", "Transcriptome Analysis", "We identified hb egfa as a secreted factor necessary and sufficient for spinal cord regeneration in zebrafish. To elucidate the Hb egfa mechanism of action  we perforemd single cell sequencing of WT and hb egfaKO zebrafish spinal cord. Overall design: Adult zebrafish were subjected to spinal cord transection. 2 mm rostral and caudal to the transection site were collected at 1 xxx post injury. Spinal cord of sham injured animals was collected as control. Samples were and subjected to the 10X Genomics pipeline for single cell sequencing 4 samples in total  WT 1 wpi  WT sham  hb egfaKO 1 wpi  hb egfaKO sham.", null, "pubmed:37567873", null, "danRer11 hbegfaKO sham", "GSM6586112", null, "source name:spinal cord|genotype:hbegfaKO|tissue:spinal cord|strain:Ekkwill EK|age:6 month|treatment:sham|geo loc name:missing|collection date:missing", "danRer11 hbegfaKO sham", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the Cell Ranger software v6.0.1 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/what is cell ranger Assembly: danRer11 Supplementary files format and content: h5 files for count table", "spinal cord", null, "Samples were processed following the 10X Chromium platform following the manufacturer\u2019s guidelines  using 10X Single Cell 3\u2019 v3 chemistry 10X Genomics  Pleasanton  USA.", null, "genotype:hbegfaKO|tissue:spinal cord|strain:Ekkwill EK|age:6 month|treatment:sham", "GSM6586112", "GSM6586112: danRer11 hbegfaKO sham; Danio rerio; RNA Seq", "GSM6586112 r1", "GSM6586112", "1", "Samples were processed following the 10X Chromium platform following the manufacturer's guidelines  using 10X Single Cell three prime v3 chemistry 10X Genomics  Pleasanton  USA.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "DNBSEQ", "DNBSEQ-G400", null, "SRP397443", null, null, "KP_VC_sample1_S1_L001_R1_001.fastq.gz KP_VC_sample1_S1_L001_R2_001.fastq.gz", "fastq fastq", 50009919813.0, 420251427.0, "GSM6586112 r1", "0:28 1:91", "A:13721824199;C:11296818044;G:11807686254;T:13179637678;N:3953638", 28, 91, null, null, 13721824199, 11296818044, 11807686254, 13179637678, 3953638, "SRX17592665", "SRS15130330", "SRA1500311", "Cell biology, Duke University", "Poss, Cell Biology, Duke University", 2, 0.01254, 0.83945, 0.00371, 0.16358, 0.98378, 0.80728, 0.25895, 0.57907, 28, 91, "T", "B", "sc-like readlen", "bgi", "bgi", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2022-09-15", "Adult", "Adult", "Spinal Cord", "Nervous System"], [74357, "SRR23690175", "SRX19552676", "SRS16937434", "SRP425371", "PRJNA940435", "sox1a:eGFP transgenic line and single cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons", "GSE226494", "Transcriptome Analysis", "The Sox family of transcription factors plays a crucial role in the development of the vertebrate nervous system. In the zebrafish embryo  sox1 genes are expressed in neural progenitor cells and neurons of the ventral spinal cord. We recently reported that the loss of function of sox1a and sox1b results in a significant decrease in a subtype of V2 neurons  called V2s  in zebrafish. Here a single cell RNA sequencing scRNA Seq approach was used to analyse the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four different time points during the development of an earlier established Tgsox1a:EGFP line. In addition to the sox1a expressing neurons described previously  we found that this gene is also expressed in late developing intraspinal serotonin neurons ISNs. Developmental trajectory analysis of single cell data and morpholino knockdown of lateral floor plate LFP cells expressing the nkx2.9 gene suggest that ISNs are generated from LFP progenitor cells. Pharmacological inhibition of the Notch signalling pathway demonstrates the necessity of this pathway for the development of LFP progenitor cells into ISN populations. Our results confirm that the zebrafish LFP is a progenitor domain from which ISN neurons are generated in addition to the previously described KA\" and V3. Overall design: The trunk and tail of Tgsox1a:eGFP lines were and dissected at 1  2 dpf 3 dpf and 5 dpf. The tissue  was dissociated and GFP positive cells were sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation  cDNA synthesis by 10X Genomics. The reads were aligned to zebrafish genom GRChZ 11 and ensemble version 105 was used for gene annotation and assigning reads to genes. Genomics Kit  and Sequencing done by Illumina.", null, "pubmed:37529101", null, "sox1a 5dpf", "GSM7077910", null, "source name:spinal cord|tissue:spinal cord|genotype:Tgsox1a:eGFP|time point:5dpf|geo loc name:missing|collection date:missing", "sox1a 5dpf", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 104 by using STAR. The BAM files were as input for Cell Ranger 10X genmoics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "spinal cord", "No treatment done.", "Cells from zebrafish spinla cord were dissociated  and sorted by FACS based on the reporter line. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH\u00a07.5  at 28\u00a0\u00b0C \u00b11\u00a0\u00b0C in groups of 20 animals per 2.8\u00a0L.\u00a0", "tissue:spinal cord|genotype:Tgsox1a:eGFP|time point:5dpf", "GSM7077910", "GSM7077910: sox1a 5dpf; Danio rerio; RNA Seq", "GSM7077910 r1", "GSM7077910", "1", "Cells from zebrafish spinla cord were dissociated  and sorted by FACS based on the reporter line. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP425371", null, "loader:fastq load.py", "L100349_Track-139094_R1.fastq.gz L100349_Track-139094_R2.fastq.gz", "fastq fastq", 78287518950.0, 387561975.0, "GSM7077910 r1", "0:101 1:101", "A:24402003655;C:12853163240;G:13012950072;T:28016227491;N:3174492", 101, 101, null, null, 24402003655, 12853163240, 13012950072, 28016227491, 3174492, "SRX19552676", "SRS16937434", "SRA1601794", "Rastegar, Institute of Biological and Chemical Systems-Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT)", "Rastegar, Institute of Biological and Chemical Systems-Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT)", 2, 0.0, 0.8342, 0.0, 0.22003, 1.0, 0.75797, null, 0.52045, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-03-02", "Larval", "Larval", "Spinal Cord", "Nervous System"], [74358, "SRR23690176", "SRX19552675", "SRS16937433", "SRP425371", "PRJNA940435", "sox1a:eGFP transgenic line and single cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons", "GSE226494", "Transcriptome Analysis", "The Sox family of transcription factors plays a crucial role in the development of the vertebrate nervous system. In the zebrafish embryo  sox1 genes are expressed in neural progenitor cells and neurons of the ventral spinal cord. We recently reported that the loss of function of sox1a and sox1b results in a significant decrease in a subtype of V2 neurons  called V2s  in zebrafish. Here a single cell RNA sequencing scRNA Seq approach was used to analyse the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four different time points during the development of an earlier established Tgsox1a:EGFP line. In addition to the sox1a expressing neurons described previously  we found that this gene is also expressed in late developing intraspinal serotonin neurons ISNs. Developmental trajectory analysis of single cell data and morpholino knockdown of lateral floor plate LFP cells expressing the nkx2.9 gene suggest that ISNs are generated from LFP progenitor cells. Pharmacological inhibition of the Notch signalling pathway demonstrates the necessity of this pathway for the development of LFP progenitor cells into ISN populations. Our results confirm that the zebrafish LFP is a progenitor domain from which ISN neurons are generated in addition to the previously described KA\" and V3. Overall design: The trunk and tail of Tgsox1a:eGFP lines were and dissected at 1  2 dpf 3 dpf and 5 dpf. The tissue  was dissociated and GFP positive cells were sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation  cDNA synthesis by 10X Genomics. The reads were aligned to zebrafish genom GRChZ 11 and ensemble version 105 was used for gene annotation and assigning reads to genes. Genomics Kit  and Sequencing done by Illumina.", null, "pubmed:37529101", null, "sox1a 3dpf", "GSM7077909", null, "source name:spinal cord|tissue:spinal cord|genotype:Tgsox1a:eGFP|time point:3dpf|geo loc name:missing|collection date:missing", "sox1a 3dpf", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 104 by using STAR. The BAM files were as input for Cell Ranger 10X genmoics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "spinal cord", "No treatment done.", "Cells from zebrafish spinla cord were dissociated  and sorted by FACS based on the reporter line. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH\u00a07.5  at 28\u00a0\u00b0C \u00b11\u00a0\u00b0C in groups of 20 animals per 2.8\u00a0L.\u00a0", "tissue:spinal cord|genotype:Tgsox1a:eGFP|time point:3dpf", "GSM7077909", "GSM7077909: sox1a 3dpf; Danio rerio; RNA Seq", "GSM7077909 r1", "GSM7077909", "1", "Cells from zebrafish spinla cord were dissociated  and sorted by FACS based on the reporter line. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP425371", null, "assembly:GRCz11|intentional duplicate|dangling references:treat as unmapped", "3dpf_possorted_genome_bam.bam", "10X Genomics bam file", 6388654202.0, 63254002.0, "GSM7077909 r1", "0:101", "A:1852098464;C:1353183909;G:1514805659;T:1668367060;N:199110", 101, null, null, null, 1852098464, 1353183909, 1514805659, 1668367060, 199110, "SRX19552675", "SRS16937433", "SRA1601794", "Rastegar, Institute of Biological and Chemical Systems-Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT)", "Rastegar, Institute of Biological and Chemical Systems-Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT)", 1, 0.90517, null, 0.25848, null, 0.77025, null, 0.56264, null, 101, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-03-02", "Larval", "Larval", "Spinal Cord", "Nervous System"], [74359, "SRR23690177", "SRX19552674", "SRS16937432", "SRP425371", "PRJNA940435", "sox1a:eGFP transgenic line and single cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons", "GSE226494", "Transcriptome Analysis", "The Sox family of transcription factors plays a crucial role in the development of the vertebrate nervous system. In the zebrafish embryo  sox1 genes are expressed in neural progenitor cells and neurons of the ventral spinal cord. We recently reported that the loss of function of sox1a and sox1b results in a significant decrease in a subtype of V2 neurons  called V2s  in zebrafish. Here a single cell RNA sequencing scRNA Seq approach was used to analyse the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four different time points during the development of an earlier established Tgsox1a:EGFP line. In addition to the sox1a expressing neurons described previously  we found that this gene is also expressed in late developing intraspinal serotonin neurons ISNs. Developmental trajectory analysis of single cell data and morpholino knockdown of lateral floor plate LFP cells expressing the nkx2.9 gene suggest that ISNs are generated from LFP progenitor cells. Pharmacological inhibition of the Notch signalling pathway demonstrates the necessity of this pathway for the development of LFP progenitor cells into ISN populations. Our results confirm that the zebrafish LFP is a progenitor domain from which ISN neurons are generated in addition to the previously described KA\" and V3. Overall design: The trunk and tail of Tgsox1a:eGFP lines were and dissected at 1  2 dpf 3 dpf and 5 dpf. The tissue  was dissociated and GFP positive cells were sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation  cDNA synthesis by 10X Genomics. The reads were aligned to zebrafish genom GRChZ 11 and ensemble version 105 was used for gene annotation and assigning reads to genes. Genomics Kit  and Sequencing done by Illumina.", null, "pubmed:37529101", null, "sox1a 2dpf", "GSM7077908", null, "source name:spinal cord|tissue:spinal cord|genotype:Tgsox1a:eGFP|time point:2dpf|geo loc name:missing|collection date:missing", "sox1a 2dpf", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 104 by using STAR. The BAM files were as input for Cell Ranger 10X genmoics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "spinal cord", "No treatment done.", "Cells from zebrafish spinla cord were dissociated  and sorted by FACS based on the reporter line. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH\u00a07.5  at 28\u00a0\u00b0C \u00b11\u00a0\u00b0C in groups of 20 animals per 2.8\u00a0L.\u00a0", "tissue:spinal cord|genotype:Tgsox1a:eGFP|time point:2dpf", "GSM7077908", "GSM7077908: sox1a 2dpf; Danio rerio; RNA Seq", "GSM7077908 r1", "GSM7077908", "1", "Cells from zebrafish spinla cord were dissociated  and sorted by FACS based on the reporter line. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP425371", null, "assembly:GRCz11|intentional duplicate|dangling references:treat as unmapped", "2dpf_possorted_genome_bam.bam", "10X Genomics bam file", 6112999144.0, 60524744.0, "GSM7077908 r1", "0:101", "A:1707014148;C:1361654515;G:1549767034;T:1494373967;N:189480", 101, null, null, null, 1707014148, 1361654515, 1549767034, 1494373967, 189480, "SRX19552674", "SRS16937432", "SRA1601794", "Rastegar, Institute of Biological and Chemical Systems-Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT)", "Rastegar, Institute of Biological and Chemical Systems-Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT)", 1, 0.90304, null, 0.26514, null, 0.79117, null, 0.59637, null, 101, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-03-02", "Hatching", "Embryo", "Spinal Cord", "Nervous System"], [74360, "SRR23690178", "SRX19552673", "SRS16937431", "SRP425371", "PRJNA940435", "sox1a:eGFP transgenic line and single cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons", "GSE226494", "Transcriptome Analysis", "The Sox family of transcription factors plays a crucial role in the development of the vertebrate nervous system. In the zebrafish embryo  sox1 genes are expressed in neural progenitor cells and neurons of the ventral spinal cord. We recently reported that the loss of function of sox1a and sox1b results in a significant decrease in a subtype of V2 neurons  called V2s  in zebrafish. Here a single cell RNA sequencing scRNA Seq approach was used to analyse the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four different time points during the development of an earlier established Tgsox1a:EGFP line. In addition to the sox1a expressing neurons described previously  we found that this gene is also expressed in late developing intraspinal serotonin neurons ISNs. Developmental trajectory analysis of single cell data and morpholino knockdown of lateral floor plate LFP cells expressing the nkx2.9 gene suggest that ISNs are generated from LFP progenitor cells. Pharmacological inhibition of the Notch signalling pathway demonstrates the necessity of this pathway for the development of LFP progenitor cells into ISN populations. Our results confirm that the zebrafish LFP is a progenitor domain from which ISN neurons are generated in addition to the previously described KA\" and V3. Overall design: The trunk and tail of Tgsox1a:eGFP lines were and dissected at 1  2 dpf 3 dpf and 5 dpf. The tissue  was dissociated and GFP positive cells were sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation  cDNA synthesis by 10X Genomics. The reads were aligned to zebrafish genom GRChZ 11 and ensemble version 105 was used for gene annotation and assigning reads to genes. Genomics Kit  and Sequencing done by Illumina.", null, "pubmed:37529101", null, "sox1a 1dpf", "GSM7077907", null, "source name:spinal cord|tissue:spinal cord|genotype:Tgsox1a:eGFP|time point:1dpf|geo loc name:missing|collection date:missing", "sox1a 1dpf", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 104 by using STAR. The BAM files were as input for Cell Ranger 10X genmoics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "spinal cord", "No treatment done.", "Cells from zebrafish spinla cord were dissociated  and sorted by FACS based on the reporter line. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH\u00a07.5  at 28\u00a0\u00b0C \u00b11\u00a0\u00b0C in groups of 20 animals per 2.8\u00a0L.\u00a0", "tissue:spinal cord|genotype:Tgsox1a:eGFP|time point:1dpf", "GSM7077907", "GSM7077907: sox1a 1dpf; Danio rerio; RNA Seq", "GSM7077907 r1", "GSM7077907", "1", "Cells from zebrafish spinla cord were dissociated  and sorted by FACS based on the reporter line. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP425371", null, "intentional duplicate", "1dpf_possorted_genome_bam.bam", "10X Genomics bam file", 41744068408.0, 413307608.0, "GSM7077907 r1", "0:101", "A:11577495181;C:9696023908;G:10077115759;T:10392411778;N:1021782", 101, null, null, null, 11577495181, 9696023908, 10077115759, 10392411778, 1021782, "SRX19552673", "SRS16937431", "SRA1601794", "Rastegar, Institute of Biological and Chemical Systems-Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT)", "Rastegar, Institute of Biological and Chemical Systems-Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT)", 1, 0.93863, null, 0.11011, null, 0.8046, null, 0.47593, null, 101, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-03-02", "Pharyngula", "Embryo", "Spinal Cord", "Nervous System"], [76472, "SRR24974217", "SRX20731837", "SRS18022738", "SRP444940", "PRJNA985790", "Single cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair", "GSE235395", "Transcriptome Analysis", "Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here  we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory xxx post injury. In addition  a transient population of injury responsive neurons iNeurons show elevated plasticity between 1 and 3 xxx post injury. We found iNeurons are injury surviving neurons that acquire a neuroblast like gene expression xxx post injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1  3  and 6 xxx post injury wpi for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point  and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury induced signals at 1 wpi  neuronal and glial regeneration at 3 wpi  and cellular remodeling at late stages of regeneration at 6 wpi Mokalled et al.  2016. Isolated nuclei were sequenced using 10x genomics platform three prime v3.1 chemistry and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation Lawson et al.  2020; Matson et al.  2018. Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA  respectively McGinnis et al.  2019; Yang et al.  2020. A second round of filtering is performed by thresholding the covariates such as number of counts  genes and fraction of counts from mitochondrial genes per barcode. A total of 58 973 nuclei was obtained for downstream analysis using the Seurat package .", null, "pubmed:39147780", null, "EKAB Spinal Cord 6 wpi rep2", "GSM7501739", null, "source name:Spinal Cord|tissue:Spinal Cord|genotype:Wild type|treatment:Injured|geo loc name:missing|collection date:missing", "EKAB Spinal Cord 6 wpi rep2", "post sequencing  the Illumina output was processed using the CellRanger v6.0.0 recommended pipeline to generate gene barcode count matrices. A custom reference genome was made with the \u201ccellranger mkref\u201d command  using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build https://useast.ensembl.org/Danio rerio/Info/Index  and the sorted Gene Transfer Format file v4.3.2 from the improved zebrafish transcriptome annotation Lawson et al.  2020 Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then  the \u201ccellranger count\u201d pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were included during the alignment. The filtered gene barcode count matrices generated by \u201ccellranger count\u201d was used for downstream analysis. All the datasets were integrated and analyzed using Seurat v4.1.1 package with R v4.2.1 R Core Team  2018; Stuart et al.  2019.  Each sample count matrix was filtered for genes that were expressed in at least 3 cells and cells expressing at least 200 genes  followed by cell quality assessment using commonly used QC matrixes Ilicic et al.  2016. Cells having a unique number of genes between 200 to 4000 and a mitochondrial gene percentage <5 were used for downstream processing. Each dataset was independently normalized and scaled using the \u201cSCTransform\u201d function  which is an improved method for normalization  that performs a variance stabilizing transformation using negative binomial regression Hafemeister and Satija  2019. Standard integration workflow of Seurat was used to identify shared sources of variation across experiments as well as mutual nearest neighbors Butler et al.  2018; Haghverdi et al.  2018. Integration features were selected based on the top 4000 highly variable features using \u201cSelectIntegrationFeatures\u201d function nfeatures = 4000  which was used as input for the \u201canchor.features\u201d argument of the \u201cFindIntegrationAnchors\u201d function. PCA analysis was performed on the 4000 variable features and the top 50 principal components selected based on the elbow plot heuristic  which measures the contribution of variation in each component. These 50 principal components were used in \u201cFindNeighbors\u201d and \u201cFindClusters\u201d functions to perform graph based clustering on a shared nearest neighbor graph Levine et al.  2015; Xu and Su  2015. Louvain algorithm was used for modularity optimization in clustering the cells using \u201cFindClusters\u201d function. The resolution parameter res = 0.5 that determines the granularity of the clustering was selected by visually inspecting clusters with resolutions ranging 0.1   2.0 as well as clustree graphs Zappia and Oshlack  2018. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 50 principal components and visualize the data using \u201cRunUMAP\u201d function Becht et al.  2018. Data was graphed using different plot functions  such as \u201cDimPlot\u201d  \u201cVlnPlot\u201d  \u201cFeaturePlot\u201d  \u201cDotplot\u201d and \u201cDoHeatmap\u201d  to view the cell cluster identity and marker gene expression. Cell proportion data was extracted using \u201ctable\u201d and \u201cprop.table\u201d functions. Differential gene expression for individual cluster was identified using Wilcoxon rank sum test in the \u201cFindAllMarkers\u201d function. Marker genes detected in at least 25% of the clustered cells and with a logFC threshold of 0.25 were selected. The clusters of interest were subclustered using the \u201csubset\u201d function for subcluster analysis. The subset was again normalized and scaled using \u201cSCTransform\u201d function with glmGamPoi method Ahlmann Eltze and Huber  2021. Fifty principal components were used  and the resolution parameter was set to 0.5. Further downstream analysis was done as described above for the integrated analysis. Assembly: GRCz11 Supplementary files format and content: Compressed tab separated files and matrix files", "Spinal Cord", null, "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3\u2019 Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell 3\u2019 Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer\u2019s protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "tissue:Spinal Cord|genotype:Wild type|treatment:Injured", "GSM7501739", "GSM7501739: EKAB Spinal Cord 6 wpi rep2; Danio rerio; RNA Seq", "GSM7501739 r1", "GSM7501739", "1", "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell three prime Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell three prime Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP444940", null, "loader:fastq load.py", "MLAG-EKAB_6wpi-EKAB_6wpi_060222-lib1_S1_L004_R2_001.fastq.gz MLAG-EKAB_6wpi-EKAB_6wpi_060222-lib1_S1_L004_R1_001.fastq.gz", "fastq fastq", 105391161150.0, 592085175.0, "GSM7501739 r1", "0:28 1:150", "A:35062246811;C:19059207172;G:22616069136;T:28650968975;N:2669056", 28, 150, null, null, 35062246811, 19059207172, 22616069136, 28650968975, 2669056, "SRX20731837", "SRS18022738", "SRA1659265", "Mokalled Lab, Developmental Biology, Washington University in St. Louis", "Developmental Biology, Washington University in St. Louis", 2, 0.00879, 0.72202, 0.00538, 0.51574, 0.99452, 0.83798, 0.28129, 0.53924, 28, 150, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-06-20", "Undetermined", "Adult", "Spinal Cord", "Nervous System"], [76473, "SRR24974218", "SRX20731836", "SRS18022737", "SRP444940", "PRJNA985790", "Single cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair", "GSE235395", "Transcriptome Analysis", "Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here  we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory xxx post injury. In addition  a transient population of injury responsive neurons iNeurons show elevated plasticity between 1 and 3 xxx post injury. We found iNeurons are injury surviving neurons that acquire a neuroblast like gene expression xxx post injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1  3  and 6 xxx post injury wpi for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point  and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury induced signals at 1 wpi  neuronal and glial regeneration at 3 wpi  and cellular remodeling at late stages of regeneration at 6 wpi Mokalled et al.  2016. Isolated nuclei were sequenced using 10x genomics platform three prime v3.1 chemistry and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation Lawson et al.  2020; Matson et al.  2018. Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA  respectively McGinnis et al.  2019; Yang et al.  2020. A second round of filtering is performed by thresholding the covariates such as number of counts  genes and fraction of counts from mitochondrial genes per barcode. A total of 58 973 nuclei was obtained for downstream analysis using the Seurat package .", null, "pubmed:39147780", null, "EKAB Spinal Cord 6 wpi rep1", "GSM7501738", null, "source name:Spinal Cord|tissue:Spinal Cord|genotype:Wild type|treatment:Injured|geo loc name:missing|collection date:missing", "EKAB Spinal Cord 6 wpi rep1", "post sequencing  the Illumina output was processed using the CellRanger v6.0.0 recommended pipeline to generate gene barcode count matrices. A custom reference genome was made with the \u201ccellranger mkref\u201d command  using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build https://useast.ensembl.org/Danio rerio/Info/Index  and the sorted Gene Transfer Format file v4.3.2 from the improved zebrafish transcriptome annotation Lawson et al.  2020 Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then  the \u201ccellranger count\u201d pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were included during the alignment. The filtered gene barcode count matrices generated by \u201ccellranger count\u201d was used for downstream analysis. All the datasets were integrated and analyzed using Seurat v4.1.1 package with R v4.2.1 R Core Team  2018; Stuart et al.  2019.  Each sample count matrix was filtered for genes that were expressed in at least 3 cells and cells expressing at least 200 genes  followed by cell quality assessment using commonly used QC matrixes Ilicic et al.  2016. Cells having a unique number of genes between 200 to 4000 and a mitochondrial gene percentage <5 were used for downstream processing. Each dataset was independently normalized and scaled using the \u201cSCTransform\u201d function  which is an improved method for normalization  that performs a variance stabilizing transformation using negative binomial regression Hafemeister and Satija  2019. Standard integration workflow of Seurat was used to identify shared sources of variation across experiments as well as mutual nearest neighbors Butler et al.  2018; Haghverdi et al.  2018. Integration features were selected based on the top 4000 highly variable features using \u201cSelectIntegrationFeatures\u201d function nfeatures = 4000  which was used as input for the \u201canchor.features\u201d argument of the \u201cFindIntegrationAnchors\u201d function. PCA analysis was performed on the 4000 variable features and the top 50 principal components selected based on the elbow plot heuristic  which measures the contribution of variation in each component. These 50 principal components were used in \u201cFindNeighbors\u201d and \u201cFindClusters\u201d functions to perform graph based clustering on a shared nearest neighbor graph Levine et al.  2015; Xu and Su  2015. Louvain algorithm was used for modularity optimization in clustering the cells using \u201cFindClusters\u201d function. The resolution parameter res = 0.5 that determines the granularity of the clustering was selected by visually inspecting clusters with resolutions ranging 0.1   2.0 as well as clustree graphs Zappia and Oshlack  2018. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 50 principal components and visualize the data using \u201cRunUMAP\u201d function Becht et al.  2018. Data was graphed using different plot functions  such as \u201cDimPlot\u201d  \u201cVlnPlot\u201d  \u201cFeaturePlot\u201d  \u201cDotplot\u201d and \u201cDoHeatmap\u201d  to view the cell cluster identity and marker gene expression. Cell proportion data was extracted using \u201ctable\u201d and \u201cprop.table\u201d functions. Differential gene expression for individual cluster was identified using Wilcoxon rank sum test in the \u201cFindAllMarkers\u201d function. Marker genes detected in at least 25% of the clustered cells and with a logFC threshold of 0.25 were selected. The clusters of interest were subclustered using the \u201csubset\u201d function for subcluster analysis. The subset was again normalized and scaled using \u201cSCTransform\u201d function with glmGamPoi method Ahlmann Eltze and Huber  2021. Fifty principal components were used  and the resolution parameter was set to 0.5. Further downstream analysis was done as described above for the integrated analysis. Assembly: GRCz11 Supplementary files format and content: Compressed tab separated files and matrix files", "Spinal Cord", null, "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3\u2019 Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell 3\u2019 Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer\u2019s protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "tissue:Spinal Cord|genotype:Wild type|treatment:Injured", "GSM7501738", "GSM7501738: EKAB Spinal Cord 6 wpi rep1; Danio rerio; RNA Seq", "GSM7501738 r1", "GSM7501738", "1", "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell three prime Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell three prime Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP444940", null, "loader:fastq load.py", "MLAG-6wpi-EKAB-6wpi_EKAB_061621-lib1_S1_L003_R2_001.fastq.gz MLAG-6wpi-EKAB-6wpi_EKAB_061621-lib1_S1_L003_R1_001.fastq.gz", "fastq fastq", 84895623668.0, 476941706.0, "GSM7501738 r1", "0:28 1:150", "A:26734879880;C:16050029642;G:18461921593;T:23647200445;N:1592108", 28, 150, null, null, 26734879880, 16050029642, 18461921593, 23647200445, 1592108, "SRX20731836", "SRS18022737", "SRA1659265", "Mokalled Lab, Developmental Biology, Washington University in St. Louis", "Developmental Biology, Washington University in St. Louis", 2, 0.01259, 0.78757, 0.00729, 0.49153, 0.99511, 0.83721, 0.27956, 0.64396, 28, 150, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-06-20", "Undetermined", "Adult", "Spinal Cord", "Nervous System"], [76474, "SRR24974219", "SRX20731835", "SRS18022736", "SRP444940", "PRJNA985790", "Single cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair", "GSE235395", "Transcriptome Analysis", "Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here  we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory xxx post injury. In addition  a transient population of injury responsive neurons iNeurons show elevated plasticity between 1 and 3 xxx post injury. We found iNeurons are injury surviving neurons that acquire a neuroblast like gene expression xxx post injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1  3  and 6 xxx post injury wpi for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point  and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury induced signals at 1 wpi  neuronal and glial regeneration at 3 wpi  and cellular remodeling at late stages of regeneration at 6 wpi Mokalled et al.  2016. Isolated nuclei were sequenced using 10x genomics platform three prime v3.1 chemistry and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation Lawson et al.  2020; Matson et al.  2018. Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA  respectively McGinnis et al.  2019; Yang et al.  2020. A second round of filtering is performed by thresholding the covariates such as number of counts  genes and fraction of counts from mitochondrial genes per barcode. A total of 58 973 nuclei was obtained for downstream analysis using the Seurat package .", null, "pubmed:39147780", null, "EKAB Spinal Cord 3 wpi rep2", "GSM7501737", null, "source name:Spinal Cord|tissue:Spinal Cord|genotype:Wild type|treatment:Injured|geo loc name:missing|collection date:missing", "EKAB Spinal Cord 3 wpi rep2", "post sequencing  the Illumina output was processed using the CellRanger v6.0.0 recommended pipeline to generate gene barcode count matrices. A custom reference genome was made with the \u201ccellranger mkref\u201d command  using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build https://useast.ensembl.org/Danio rerio/Info/Index  and the sorted Gene Transfer Format file v4.3.2 from the improved zebrafish transcriptome annotation Lawson et al.  2020 Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then  the \u201ccellranger count\u201d pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were included during the alignment. The filtered gene barcode count matrices generated by \u201ccellranger count\u201d was used for downstream analysis. All the datasets were integrated and analyzed using Seurat v4.1.1 package with R v4.2.1 R Core Team  2018; Stuart et al.  2019.  Each sample count matrix was filtered for genes that were expressed in at least 3 cells and cells expressing at least 200 genes  followed by cell quality assessment using commonly used QC matrixes Ilicic et al.  2016. Cells having a unique number of genes between 200 to 4000 and a mitochondrial gene percentage <5 were used for downstream processing. Each dataset was independently normalized and scaled using the \u201cSCTransform\u201d function  which is an improved method for normalization  that performs a variance stabilizing transformation using negative binomial regression Hafemeister and Satija  2019. Standard integration workflow of Seurat was used to identify shared sources of variation across experiments as well as mutual nearest neighbors Butler et al.  2018; Haghverdi et al.  2018. Integration features were selected based on the top 4000 highly variable features using \u201cSelectIntegrationFeatures\u201d function nfeatures = 4000  which was used as input for the \u201canchor.features\u201d argument of the \u201cFindIntegrationAnchors\u201d function. PCA analysis was performed on the 4000 variable features and the top 50 principal components selected based on the elbow plot heuristic  which measures the contribution of variation in each component. These 50 principal components were used in \u201cFindNeighbors\u201d and \u201cFindClusters\u201d functions to perform graph based clustering on a shared nearest neighbor graph Levine et al.  2015; Xu and Su  2015. Louvain algorithm was used for modularity optimization in clustering the cells using \u201cFindClusters\u201d function. The resolution parameter res = 0.5 that determines the granularity of the clustering was selected by visually inspecting clusters with resolutions ranging 0.1   2.0 as well as clustree graphs Zappia and Oshlack  2018. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 50 principal components and visualize the data using \u201cRunUMAP\u201d function Becht et al.  2018. Data was graphed using different plot functions  such as \u201cDimPlot\u201d  \u201cVlnPlot\u201d  \u201cFeaturePlot\u201d  \u201cDotplot\u201d and \u201cDoHeatmap\u201d  to view the cell cluster identity and marker gene expression. Cell proportion data was extracted using \u201ctable\u201d and \u201cprop.table\u201d functions. Differential gene expression for individual cluster was identified using Wilcoxon rank sum test in the \u201cFindAllMarkers\u201d function. Marker genes detected in at least 25% of the clustered cells and with a logFC threshold of 0.25 were selected. The clusters of interest were subclustered using the \u201csubset\u201d function for subcluster analysis. The subset was again normalized and scaled using \u201cSCTransform\u201d function with glmGamPoi method Ahlmann Eltze and Huber  2021. Fifty principal components were used  and the resolution parameter was set to 0.5. Further downstream analysis was done as described above for the integrated analysis. Assembly: GRCz11 Supplementary files format and content: Compressed tab separated files and matrix files", "Spinal Cord", null, "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3\u2019 Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell 3\u2019 Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer\u2019s protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "tissue:Spinal Cord|genotype:Wild type|treatment:Injured", "GSM7501737", "GSM7501737: EKAB Spinal Cord 3 wpi rep2; Danio rerio; RNA Seq", "GSM7501737 r1", "GSM7501737", "1", "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell three prime Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell three prime Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP444940", null, "loader:fastq load.py", "MLAG-EKAB_3wpi-EKAB_3wpi_060222-lib1_S2_L004_R2_001.fastq.gz MLAG-EKAB_3wpi-EKAB_3wpi_060222-lib1_S2_L004_R1_001.fastq.gz", "fastq fastq", 101322905824.0, 569229808.0, "GSM7501737 r1", "0:28 1:150", "A:33529740337;C:18508795541;G:22218128467;T:27063600389;N:2641090", 28, 150, null, null, 33529740337, 18508795541, 22218128467, 27063600389, 2641090, "SRX20731835", "SRS18022736", "SRA1659265", "Mokalled Lab, Developmental Biology, Washington University in St. Louis", "Developmental Biology, Washington University in St. Louis", 2, 0.01016, 0.72116, 0.00605, 0.48692, 0.99462, 0.83666, 0.28172, 0.53814, 28, 150, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-06-20", "Undetermined", "Adult", "Spinal Cord", "Nervous System"], [76475, "SRR24974220", "SRX20731834", "SRS18022735", "SRP444940", "PRJNA985790", "Single cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair", "GSE235395", "Transcriptome Analysis", "Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here  we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory xxx post injury. In addition  a transient population of injury responsive neurons iNeurons show elevated plasticity between 1 and 3 xxx post injury. We found iNeurons are injury surviving neurons that acquire a neuroblast like gene expression xxx post injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1  3  and 6 xxx post injury wpi for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point  and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury induced signals at 1 wpi  neuronal and glial regeneration at 3 wpi  and cellular remodeling at late stages of regeneration at 6 wpi Mokalled et al.  2016. Isolated nuclei were sequenced using 10x genomics platform three prime v3.1 chemistry and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation Lawson et al.  2020; Matson et al.  2018. Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA  respectively McGinnis et al.  2019; Yang et al.  2020. A second round of filtering is performed by thresholding the covariates such as number of counts  genes and fraction of counts from mitochondrial genes per barcode. A total of 58 973 nuclei was obtained for downstream analysis using the Seurat package .", null, "pubmed:39147780", null, "EKAB Spinal Cord 3 wpi rep1", "GSM7501736", null, "source name:Spinal Cord|tissue:Spinal Cord|genotype:Wild type|treatment:Injured|geo loc name:missing|collection date:missing", "EKAB Spinal Cord 3 wpi rep1", "post sequencing  the Illumina output was processed using the CellRanger v6.0.0 recommended pipeline to generate gene barcode count matrices. A custom reference genome was made with the \u201ccellranger mkref\u201d command  using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build https://useast.ensembl.org/Danio rerio/Info/Index  and the sorted Gene Transfer Format file v4.3.2 from the improved zebrafish transcriptome annotation Lawson et al.  2020 Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then  the \u201ccellranger count\u201d pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were included during the alignment. The filtered gene barcode count matrices generated by \u201ccellranger count\u201d was used for downstream analysis. All the datasets were integrated and analyzed using Seurat v4.1.1 package with R v4.2.1 R Core Team  2018; Stuart et al.  2019.  Each sample count matrix was filtered for genes that were expressed in at least 3 cells and cells expressing at least 200 genes  followed by cell quality assessment using commonly used QC matrixes Ilicic et al.  2016. Cells having a unique number of genes between 200 to 4000 and a mitochondrial gene percentage <5 were used for downstream processing. Each dataset was independently normalized and scaled using the \u201cSCTransform\u201d function  which is an improved method for normalization  that performs a variance stabilizing transformation using negative binomial regression Hafemeister and Satija  2019. Standard integration workflow of Seurat was used to identify shared sources of variation across experiments as well as mutual nearest neighbors Butler et al.  2018; Haghverdi et al.  2018. Integration features were selected based on the top 4000 highly variable features using \u201cSelectIntegrationFeatures\u201d function nfeatures = 4000  which was used as input for the \u201canchor.features\u201d argument of the \u201cFindIntegrationAnchors\u201d function. PCA analysis was performed on the 4000 variable features and the top 50 principal components selected based on the elbow plot heuristic  which measures the contribution of variation in each component. These 50 principal components were used in \u201cFindNeighbors\u201d and \u201cFindClusters\u201d functions to perform graph based clustering on a shared nearest neighbor graph Levine et al.  2015; Xu and Su  2015. Louvain algorithm was used for modularity optimization in clustering the cells using \u201cFindClusters\u201d function. The resolution parameter res = 0.5 that determines the granularity of the clustering was selected by visually inspecting clusters with resolutions ranging 0.1   2.0 as well as clustree graphs Zappia and Oshlack  2018. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 50 principal components and visualize the data using \u201cRunUMAP\u201d function Becht et al.  2018. Data was graphed using different plot functions  such as \u201cDimPlot\u201d  \u201cVlnPlot\u201d  \u201cFeaturePlot\u201d  \u201cDotplot\u201d and \u201cDoHeatmap\u201d  to view the cell cluster identity and marker gene expression. Cell proportion data was extracted using \u201ctable\u201d and \u201cprop.table\u201d functions. Differential gene expression for individual cluster was identified using Wilcoxon rank sum test in the \u201cFindAllMarkers\u201d function. Marker genes detected in at least 25% of the clustered cells and with a logFC threshold of 0.25 were selected. The clusters of interest were subclustered using the \u201csubset\u201d function for subcluster analysis. The subset was again normalized and scaled using \u201cSCTransform\u201d function with glmGamPoi method Ahlmann Eltze and Huber  2021. Fifty principal components were used  and the resolution parameter was set to 0.5. Further downstream analysis was done as described above for the integrated analysis. Assembly: GRCz11 Supplementary files format and content: Compressed tab separated files and matrix files", "Spinal Cord", null, "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3\u2019 Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell 3\u2019 Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer\u2019s protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "tissue:Spinal Cord|genotype:Wild type|treatment:Injured", "GSM7501736", "GSM7501736: EKAB Spinal Cord 3 wpi rep1; Danio rerio; RNA Seq", "GSM7501736 r1", "GSM7501736", "1", "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell three prime Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell three prime Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP444940", null, "loader:fastq load.py", "MLAG-3wpi-EKAB-3wpi_EKAB_081121-lib1_S1_L003_R1_001.fastq.gz MLAG-3wpi-EKAB-3wpi_EKAB_081121-lib1_S1_L003_R2_001.fastq.gz", "fastq fastq", 105936769400.0, 529683847.0, "GSM7501736 r1", "0:50 1:150", "A:35005213053;C:19468074725;G:21726475457;T:29732821194;N:4184971", 50, 150, null, null, 35005213053, 19468074725, 21726475457, 29732821194, 4184971, "SRX20731834", "SRS18022735", "SRA1659265", "Mokalled Lab, Developmental Biology, Washington University in St. Louis", "Developmental Biology, Washington University in St. Louis", 2, 0.0, 0.74755, 0.0, 0.45633, 1.0, 0.81667, null, 0.52945, 50, 150, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-06-20", "Undetermined", "Adult", "Spinal Cord", "Nervous System"], [76476, "SRR24974221", "SRX20731833", "SRS18022734", "SRP444940", "PRJNA985790", "Single cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair", "GSE235395", "Transcriptome Analysis", "Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here  we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory xxx post injury. In addition  a transient population of injury responsive neurons iNeurons show elevated plasticity between 1 and 3 xxx post injury. We found iNeurons are injury surviving neurons that acquire a neuroblast like gene expression xxx post injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1  3  and 6 xxx post injury wpi for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point  and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury induced signals at 1 wpi  neuronal and glial regeneration at 3 wpi  and cellular remodeling at late stages of regeneration at 6 wpi Mokalled et al.  2016. Isolated nuclei were sequenced using 10x genomics platform three prime v3.1 chemistry and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation Lawson et al.  2020; Matson et al.  2018. Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA  respectively McGinnis et al.  2019; Yang et al.  2020. A second round of filtering is performed by thresholding the covariates such as number of counts  genes and fraction of counts from mitochondrial genes per barcode. A total of 58 973 nuclei was obtained for downstream analysis using the Seurat package .", null, "pubmed:39147780", null, "EKAB Spinal Cord 1 wpi rep2", "GSM7501735", null, "source name:Spinal Cord|tissue:Spinal Cord|genotype:Wild type|treatment:Injured|geo loc name:missing|collection date:missing", "EKAB Spinal Cord 1 wpi rep2", "post sequencing  the Illumina output was processed using the CellRanger v6.0.0 recommended pipeline to generate gene barcode count matrices. A custom reference genome was made with the \u201ccellranger mkref\u201d command  using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build https://useast.ensembl.org/Danio rerio/Info/Index  and the sorted Gene Transfer Format file v4.3.2 from the improved zebrafish transcriptome annotation Lawson et al.  2020 Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then  the \u201ccellranger count\u201d pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were included during the alignment. The filtered gene barcode count matrices generated by \u201ccellranger count\u201d was used for downstream analysis. All the datasets were integrated and analyzed using Seurat v4.1.1 package with R v4.2.1 R Core Team  2018; Stuart et al.  2019.  Each sample count matrix was filtered for genes that were expressed in at least 3 cells and cells expressing at least 200 genes  followed by cell quality assessment using commonly used QC matrixes Ilicic et al.  2016. Cells having a unique number of genes between 200 to 4000 and a mitochondrial gene percentage <5 were used for downstream processing. Each dataset was independently normalized and scaled using the \u201cSCTransform\u201d function  which is an improved method for normalization  that performs a variance stabilizing transformation using negative binomial regression Hafemeister and Satija  2019. Standard integration workflow of Seurat was used to identify shared sources of variation across experiments as well as mutual nearest neighbors Butler et al.  2018; Haghverdi et al.  2018. Integration features were selected based on the top 4000 highly variable features using \u201cSelectIntegrationFeatures\u201d function nfeatures = 4000  which was used as input for the \u201canchor.features\u201d argument of the \u201cFindIntegrationAnchors\u201d function. PCA analysis was performed on the 4000 variable features and the top 50 principal components selected based on the elbow plot heuristic  which measures the contribution of variation in each component. These 50 principal components were used in \u201cFindNeighbors\u201d and \u201cFindClusters\u201d functions to perform graph based clustering on a shared nearest neighbor graph Levine et al.  2015; Xu and Su  2015. Louvain algorithm was used for modularity optimization in clustering the cells using \u201cFindClusters\u201d function. The resolution parameter res = 0.5 that determines the granularity of the clustering was selected by visually inspecting clusters with resolutions ranging 0.1   2.0 as well as clustree graphs Zappia and Oshlack  2018. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 50 principal components and visualize the data using \u201cRunUMAP\u201d function Becht et al.  2018. Data was graphed using different plot functions  such as \u201cDimPlot\u201d  \u201cVlnPlot\u201d  \u201cFeaturePlot\u201d  \u201cDotplot\u201d and \u201cDoHeatmap\u201d  to view the cell cluster identity and marker gene expression. Cell proportion data was extracted using \u201ctable\u201d and \u201cprop.table\u201d functions. Differential gene expression for individual cluster was identified using Wilcoxon rank sum test in the \u201cFindAllMarkers\u201d function. Marker genes detected in at least 25% of the clustered cells and with a logFC threshold of 0.25 were selected. The clusters of interest were subclustered using the \u201csubset\u201d function for subcluster analysis. The subset was again normalized and scaled using \u201cSCTransform\u201d function with glmGamPoi method Ahlmann Eltze and Huber  2021. Fifty principal components were used  and the resolution parameter was set to 0.5. Further downstream analysis was done as described above for the integrated analysis. Assembly: GRCz11 Supplementary files format and content: Compressed tab separated files and matrix files", "Spinal Cord", null, "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3\u2019 Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell 3\u2019 Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer\u2019s protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "tissue:Spinal Cord|genotype:Wild type|treatment:Injured", "GSM7501735", "GSM7501735: EKAB Spinal Cord 1 wpi rep2; Danio rerio; RNA Seq", "GSM7501735 r1", "GSM7501735", "1", "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell three prime Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell three prime Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP444940", null, "loader:fastq load.py", "MLAG-EKAB_1wpi-EKAB_1wpi_060122-lib1_S4_L004_R1_001.fastq.gz MLAG-EKAB_1wpi-EKAB_1wpi_060122-lib1_S4_L004_R2_001.fastq.gz", "fastq fastq", 74288465184.0, 417350928.0, "GSM7501735 r1", "0:28 1:150", "A:23894773694;C:13783822381;G:16246726428;T:20361238077;N:1904604", 28, 150, null, null, 23894773694, 13783822381, 16246726428, 20361238077, 1904604, "SRX20731833", "SRS18022734", "SRA1659265", "Mokalled Lab, Developmental Biology, Washington University in St. Louis", "Developmental Biology, Washington University in St. Louis", 2, 0.01015, 0.74263, 0.00625, 0.51946, 0.99464, 0.84062, 0.29395, 0.55744, 28, 150, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-06-20", "Undetermined", "Adult", "Spinal Cord", "Nervous System"], [76477, "SRR24974222", "SRX20731832", "SRS18022733", "SRP444940", "PRJNA985790", "Single cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair", "GSE235395", "Transcriptome Analysis", "Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here  we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory xxx post injury. In addition  a transient population of injury responsive neurons iNeurons show elevated plasticity between 1 and 3 xxx post injury. We found iNeurons are injury surviving neurons that acquire a neuroblast like gene expression xxx post injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1  3  and 6 xxx post injury wpi for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point  and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury induced signals at 1 wpi  neuronal and glial regeneration at 3 wpi  and cellular remodeling at late stages of regeneration at 6 wpi Mokalled et al.  2016. Isolated nuclei were sequenced using 10x genomics platform three prime v3.1 chemistry and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation Lawson et al.  2020; Matson et al.  2018. Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA  respectively McGinnis et al.  2019; Yang et al.  2020. A second round of filtering is performed by thresholding the covariates such as number of counts  genes and fraction of counts from mitochondrial genes per barcode. A total of 58 973 nuclei was obtained for downstream analysis using the Seurat package .", null, "pubmed:39147780", null, "EKAB Spinal Cord 1 wpi rep1", "GSM7501734", null, "source name:Spinal Cord|tissue:Spinal Cord|genotype:Wild type|treatment:Injured|geo loc name:missing|collection date:missing", "EKAB Spinal Cord 1 wpi rep1", "post sequencing  the Illumina output was processed using the CellRanger v6.0.0 recommended pipeline to generate gene barcode count matrices. A custom reference genome was made with the \u201ccellranger mkref\u201d command  using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build https://useast.ensembl.org/Danio rerio/Info/Index  and the sorted Gene Transfer Format file v4.3.2 from the improved zebrafish transcriptome annotation Lawson et al.  2020 Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then  the \u201ccellranger count\u201d pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were included during the alignment. The filtered gene barcode count matrices generated by \u201ccellranger count\u201d was used for downstream analysis. All the datasets were integrated and analyzed using Seurat v4.1.1 package with R v4.2.1 R Core Team  2018; Stuart et al.  2019.  Each sample count matrix was filtered for genes that were expressed in at least 3 cells and cells expressing at least 200 genes  followed by cell quality assessment using commonly used QC matrixes Ilicic et al.  2016. Cells having a unique number of genes between 200 to 4000 and a mitochondrial gene percentage <5 were used for downstream processing. Each dataset was independently normalized and scaled using the \u201cSCTransform\u201d function  which is an improved method for normalization  that performs a variance stabilizing transformation using negative binomial regression Hafemeister and Satija  2019. Standard integration workflow of Seurat was used to identify shared sources of variation across experiments as well as mutual nearest neighbors Butler et al.  2018; Haghverdi et al.  2018. Integration features were selected based on the top 4000 highly variable features using \u201cSelectIntegrationFeatures\u201d function nfeatures = 4000  which was used as input for the \u201canchor.features\u201d argument of the \u201cFindIntegrationAnchors\u201d function. PCA analysis was performed on the 4000 variable features and the top 50 principal components selected based on the elbow plot heuristic  which measures the contribution of variation in each component. These 50 principal components were used in \u201cFindNeighbors\u201d and \u201cFindClusters\u201d functions to perform graph based clustering on a shared nearest neighbor graph Levine et al.  2015; Xu and Su  2015. Louvain algorithm was used for modularity optimization in clustering the cells using \u201cFindClusters\u201d function. The resolution parameter res = 0.5 that determines the granularity of the clustering was selected by visually inspecting clusters with resolutions ranging 0.1   2.0 as well as clustree graphs Zappia and Oshlack  2018. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 50 principal components and visualize the data using \u201cRunUMAP\u201d function Becht et al.  2018. Data was graphed using different plot functions  such as \u201cDimPlot\u201d  \u201cVlnPlot\u201d  \u201cFeaturePlot\u201d  \u201cDotplot\u201d and \u201cDoHeatmap\u201d  to view the cell cluster identity and marker gene expression. Cell proportion data was extracted using \u201ctable\u201d and \u201cprop.table\u201d functions. Differential gene expression for individual cluster was identified using Wilcoxon rank sum test in the \u201cFindAllMarkers\u201d function. Marker genes detected in at least 25% of the clustered cells and with a logFC threshold of 0.25 were selected. The clusters of interest were subclustered using the \u201csubset\u201d function for subcluster analysis. The subset was again normalized and scaled using \u201cSCTransform\u201d function with glmGamPoi method Ahlmann Eltze and Huber  2021. Fifty principal components were used  and the resolution parameter was set to 0.5. Further downstream analysis was done as described above for the integrated analysis. Assembly: GRCz11 Supplementary files format and content: Compressed tab separated files and matrix files", "Spinal Cord", null, "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3\u2019 Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell 3\u2019 Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer\u2019s protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "tissue:Spinal Cord|genotype:Wild type|treatment:Injured", "GSM7501734", "GSM7501734: EKAB Spinal Cord 1 wpi rep1; Danio rerio; RNA Seq", "GSM7501734 r1", "GSM7501734", "1", "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell three prime Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell three prime Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP444940", null, "loader:fastq load.py", "MLAG-1wpi-EKAB-1wpi_EKAB_022521_S1_L001_R1_001.fastq.gz MLAG-1wpi-EKAB-1wpi_EKAB_022521_S1_L001_R2_001.fastq.gz", "fastq fastq", 105664571464.0, 593621188.0, "GSM7501734 r1", "0:28 1:150", "A:36626846226;C:18608728346;G:22744330287;T:27679941436;N:4725169", 28, 150, null, null, 36626846226, 18608728346, 22744330287, 27679941436, 4725169, "SRX20731832", "SRS18022733", "SRA1659265", "Mokalled Lab, Developmental Biology, Washington University in St. Louis", "Developmental Biology, Washington University in St. Louis", 2, 0.01645, 0.6943, 0.01249, 0.36909, 0.9919, 0.8309, 0.33377, 0.52756, 28, 150, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-06-20", "Undetermined", "Adult", "Spinal Cord", "Nervous System"], [76478, "SRR24974223", "SRX20731831", "SRS18022732", "SRP444940", "PRJNA985790", "Single cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair", "GSE235395", "Transcriptome Analysis", "Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here  we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory xxx post injury. In addition  a transient population of injury responsive neurons iNeurons show elevated plasticity between 1 and 3 xxx post injury. We found iNeurons are injury surviving neurons that acquire a neuroblast like gene expression xxx post injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1  3  and 6 xxx post injury wpi for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point  and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury induced signals at 1 wpi  neuronal and glial regeneration at 3 wpi  and cellular remodeling at late stages of regeneration at 6 wpi Mokalled et al.  2016. Isolated nuclei were sequenced using 10x genomics platform three prime v3.1 chemistry and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation Lawson et al.  2020; Matson et al.  2018. Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA  respectively McGinnis et al.  2019; Yang et al.  2020. A second round of filtering is performed by thresholding the covariates such as number of counts  genes and fraction of counts from mitochondrial genes per barcode. A total of 58 973 nuclei was obtained for downstream analysis using the Seurat package .", null, "pubmed:39147780", null, "EKAB Spinal Cord Uninjured rep2", "GSM7501733", null, "source name:Spinal Cord|tissue:Spinal Cord|genotype:Wild type|treatment:Uninjured|geo loc name:missing|collection date:missing", "EKAB Spinal Cord Uninjured rep2", "post sequencing  the Illumina output was processed using the CellRanger v6.0.0 recommended pipeline to generate gene barcode count matrices. A custom reference genome was made with the \u201ccellranger mkref\u201d command  using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build https://useast.ensembl.org/Danio rerio/Info/Index  and the sorted Gene Transfer Format file v4.3.2 from the improved zebrafish transcriptome annotation Lawson et al.  2020 Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then  the \u201ccellranger count\u201d pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were included during the alignment. The filtered gene barcode count matrices generated by \u201ccellranger count\u201d was used for downstream analysis. All the datasets were integrated and analyzed using Seurat v4.1.1 package with R v4.2.1 R Core Team  2018; Stuart et al.  2019.  Each sample count matrix was filtered for genes that were expressed in at least 3 cells and cells expressing at least 200 genes  followed by cell quality assessment using commonly used QC matrixes Ilicic et al.  2016. Cells having a unique number of genes between 200 to 4000 and a mitochondrial gene percentage <5 were used for downstream processing. Each dataset was independently normalized and scaled using the \u201cSCTransform\u201d function  which is an improved method for normalization  that performs a variance stabilizing transformation using negative binomial regression Hafemeister and Satija  2019. Standard integration workflow of Seurat was used to identify shared sources of variation across experiments as well as mutual nearest neighbors Butler et al.  2018; Haghverdi et al.  2018. Integration features were selected based on the top 4000 highly variable features using \u201cSelectIntegrationFeatures\u201d function nfeatures = 4000  which was used as input for the \u201canchor.features\u201d argument of the \u201cFindIntegrationAnchors\u201d function. PCA analysis was performed on the 4000 variable features and the top 50 principal components selected based on the elbow plot heuristic  which measures the contribution of variation in each component. These 50 principal components were used in \u201cFindNeighbors\u201d and \u201cFindClusters\u201d functions to perform graph based clustering on a shared nearest neighbor graph Levine et al.  2015; Xu and Su  2015. Louvain algorithm was used for modularity optimization in clustering the cells using \u201cFindClusters\u201d function. The resolution parameter res = 0.5 that determines the granularity of the clustering was selected by visually inspecting clusters with resolutions ranging 0.1   2.0 as well as clustree graphs Zappia and Oshlack  2018. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 50 principal components and visualize the data using \u201cRunUMAP\u201d function Becht et al.  2018. Data was graphed using different plot functions  such as \u201cDimPlot\u201d  \u201cVlnPlot\u201d  \u201cFeaturePlot\u201d  \u201cDotplot\u201d and \u201cDoHeatmap\u201d  to view the cell cluster identity and marker gene expression. Cell proportion data was extracted using \u201ctable\u201d and \u201cprop.table\u201d functions. Differential gene expression for individual cluster was identified using Wilcoxon rank sum test in the \u201cFindAllMarkers\u201d function. Marker genes detected in at least 25% of the clustered cells and with a logFC threshold of 0.25 were selected. The clusters of interest were subclustered using the \u201csubset\u201d function for subcluster analysis. The subset was again normalized and scaled using \u201cSCTransform\u201d function with glmGamPoi method Ahlmann Eltze and Huber  2021. Fifty principal components were used  and the resolution parameter was set to 0.5. Further downstream analysis was done as described above for the integrated analysis. Assembly: GRCz11 Supplementary files format and content: Compressed tab separated files and matrix files", "Spinal Cord", null, "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3\u2019 Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell 3\u2019 Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer\u2019s protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "tissue:Spinal Cord|genotype:Wild type|treatment:Uninjured", "GSM7501733", "GSM7501733: EKAB Spinal Cord Uninjured rep2; Danio rerio; RNA Seq", "GSM7501733 r1", "GSM7501733", "1", "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell three prime Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell three prime Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP444940", null, "loader:fastq load.py", "MLAG-EKAB_Uninj-EKAB_Uninj_060122-lib1_S3_L003_R1_001.fastq.gz MLAG-EKAB_Uninj-EKAB_Uninj_060122-lib1_S3_L003_R2_001.fastq.gz", "fastq fastq", 70360166986.0, 395281837.0, "GSM7501733 r1", "0:28 1:150", "A:23061071505;C:12770839971;G:14766106153;T:19760221018;N:1928339", 28, 150, null, null, 23061071505, 12770839971, 14766106153, 19760221018, 1928339, "SRX20731831", "SRS18022732", "SRA1659265", "Mokalled Lab, Developmental Biology, Washington University in St. Louis", "Developmental Biology, Washington University in St. Louis", 2, 0.00827, 0.72918, 0.00547, 0.5217, 0.99415, 0.82402, 0.31226, 0.5218, 28, 150, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-06-20", "Undetermined", "Adult", "Spinal Cord", "Nervous System"], [76479, "SRR24974224", "SRX20731830", "SRS18022731", "SRP444940", "PRJNA985790", "Single cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair", "GSE235395", "Transcriptome Analysis", "Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here  we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory xxx post injury. In addition  a transient population of injury responsive neurons iNeurons show elevated plasticity between 1 and 3 xxx post injury. We found iNeurons are injury surviving neurons that acquire a neuroblast like gene expression xxx post injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1  3  and 6 xxx post injury wpi for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point  and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury induced signals at 1 wpi  neuronal and glial regeneration at 3 wpi  and cellular remodeling at late stages of regeneration at 6 wpi Mokalled et al.  2016. Isolated nuclei were sequenced using 10x genomics platform three prime v3.1 chemistry and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation Lawson et al.  2020; Matson et al.  2018. Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA  respectively McGinnis et al.  2019; Yang et al.  2020. A second round of filtering is performed by thresholding the covariates such as number of counts  genes and fraction of counts from mitochondrial genes per barcode. A total of 58 973 nuclei was obtained for downstream analysis using the Seurat package .", null, "pubmed:39147780", null, "EKAB Spinal Cord Uninjured rep1", "GSM7501732", null, "source name:Spinal Cord|tissue:Spinal Cord|genotype:Wild type|treatment:Uninjured|geo loc name:missing|collection date:missing", "EKAB Spinal Cord Uninjured rep1", "post sequencing  the Illumina output was processed using the CellRanger v6.0.0 recommended pipeline to generate gene barcode count matrices. A custom reference genome was made with the \u201ccellranger mkref\u201d command  using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build https://useast.ensembl.org/Danio rerio/Info/Index  and the sorted Gene Transfer Format file v4.3.2 from the improved zebrafish transcriptome annotation Lawson et al.  2020 Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then  the \u201ccellranger count\u201d pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were included during the alignment. The filtered gene barcode count matrices generated by \u201ccellranger count\u201d was used for downstream analysis. All the datasets were integrated and analyzed using Seurat v4.1.1 package with R v4.2.1 R Core Team  2018; Stuart et al.  2019.  Each sample count matrix was filtered for genes that were expressed in at least 3 cells and cells expressing at least 200 genes  followed by cell quality assessment using commonly used QC matrixes Ilicic et al.  2016. Cells having a unique number of genes between 200 to 4000 and a mitochondrial gene percentage <5 were used for downstream processing. Each dataset was independently normalized and scaled using the \u201cSCTransform\u201d function  which is an improved method for normalization  that performs a variance stabilizing transformation using negative binomial regression Hafemeister and Satija  2019. Standard integration workflow of Seurat was used to identify shared sources of variation across experiments as well as mutual nearest neighbors Butler et al.  2018; Haghverdi et al.  2018. Integration features were selected based on the top 4000 highly variable features using \u201cSelectIntegrationFeatures\u201d function nfeatures = 4000  which was used as input for the \u201canchor.features\u201d argument of the \u201cFindIntegrationAnchors\u201d function. PCA analysis was performed on the 4000 variable features and the top 50 principal components selected based on the elbow plot heuristic  which measures the contribution of variation in each component. These 50 principal components were used in \u201cFindNeighbors\u201d and \u201cFindClusters\u201d functions to perform graph based clustering on a shared nearest neighbor graph Levine et al.  2015; Xu and Su  2015. Louvain algorithm was used for modularity optimization in clustering the cells using \u201cFindClusters\u201d function. The resolution parameter res = 0.5 that determines the granularity of the clustering was selected by visually inspecting clusters with resolutions ranging 0.1   2.0 as well as clustree graphs Zappia and Oshlack  2018. Uniform Manifold Approximation and Reduction UMAP was used for non linear dimensional reduction of the first 50 principal components and visualize the data using \u201cRunUMAP\u201d function Becht et al.  2018. Data was graphed using different plot functions  such as \u201cDimPlot\u201d  \u201cVlnPlot\u201d  \u201cFeaturePlot\u201d  \u201cDotplot\u201d and \u201cDoHeatmap\u201d  to view the cell cluster identity and marker gene expression. Cell proportion data was extracted using \u201ctable\u201d and \u201cprop.table\u201d functions. Differential gene expression for individual cluster was identified using Wilcoxon rank sum test in the \u201cFindAllMarkers\u201d function. Marker genes detected in at least 25% of the clustered cells and with a logFC threshold of 0.25 were selected. The clusters of interest were subclustered using the \u201csubset\u201d function for subcluster analysis. The subset was again normalized and scaled using \u201cSCTransform\u201d function with glmGamPoi method Ahlmann Eltze and Huber  2021. Fifty principal components were used  and the resolution parameter was set to 0.5. Further downstream analysis was done as described above for the integrated analysis. Assembly: GRCz11 Supplementary files format and content: Compressed tab separated files and matrix files", "Spinal Cord", null, "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3\u2019 Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell 3\u2019 Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer\u2019s protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "tissue:Spinal Cord|genotype:Wild type|treatment:Uninjured", "GSM7501732", "GSM7501732: EKAB Spinal Cord Uninjured rep1; Danio rerio; RNA Seq", "GSM7501732 r1", "GSM7501732", "1", "For snRNA seq  30 \u00b5l of resuspension solution containing isolated nuclei at a concentration of 1000 nuclei/\u00b5l was submitted to sequencing.Two biological replicates of each timepoints at 0  1  3 and 6 wpi were used. cDNA was prepared post the GEM generation and barcoding  followed by the GEM RT reaction and bead cleanup steps.  cDNA was amplified for 11 13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell three prime Reagent Kits User Guide v3.1 Chemistry Dual Index with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform  the Chromium Next GEM Single Cell three prime Kit v3.1  16 rxns PN 1000268  Chromium Next GEM Chip G Single Cell Kit  48 rxns PN 1000120  and Dual Index Kit TT Set A  96 rxns PN 1000215 were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol KAPA Biosystems/Roche to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50 000 reads/cell was targeted for each Gene Expression Library.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP444940", null, "loader:fastq load.py", "MLAG-Uninj-EKAB-Uninj_EKAB_022521_S2_L001_R1_001.fastq.gz MLAG-Uninj-EKAB-Uninj_EKAB_022521_S2_L001_R2_001.fastq.gz", "fastq fastq", 90709334000.0, 509603000.0, "GSM7501732 r1", "0:28 1:150", "A:31112543559;C:15894186068;G:19250857481;T:24447634392;N:4112500", 28, 150, null, null, 31112543559, 15894186068, 19250857481, 24447634392, 4112500, "SRX20731830", "SRS18022731", "SRA1659265", "Mokalled Lab, Developmental Biology, Washington University in St. Louis", "Developmental Biology, Washington University in St. Louis", 2, 0.01435, 0.67925, 0.01151, 0.41035, 0.99397, 0.83936, 0.36598, 0.58394, 28, 150, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-06-20", "Undetermined", "Adult", "Spinal Cord", "Nervous System"]], "truncated": false, "filtered_table_rows_count": 67, "expanded_columns": [], "expandable_columns": [], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", 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[experiment.attributes], [experiment.library_strategy], [experiment.library_source], [experiment.library_selection], [experiment.library_layout], [experiment.platform], [experiment.instrument_model], [experiment.spot_descriptor], [experiment.study_ref], [run.title], [run.attributes], [run.filename], [run.semantic_name], [run.total_bases], [run.total_spots], [run.alias], [run.read_lengths], [run.base_counts], [run.r1_length], [run.r2_length], [run.r3_length], [run.r4_length], [run.Acount], [run.Ccount], [run.Gcount], [run.Tcount], [run.Ncount], [run.experiment], [run.pool_member], [submission.accession], [submission.srasource], [submission.bioprojectsource], [seqdetective.n_mates], [seqdetective.mapping_rate.mate1], [seqdetective.mapping_rate.mate2], [seqdetective.nofeature_rate.mate1], [seqdetective.nofeature_rate.mate2], [seqdetective.sparsity.mate1], [seqdetective.sparsity.mate2], [seqdetective.pos_strand_rate.mate1], [seqdetective.pos_strand_rate.mate2], 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