GSM9393582: Multiome, scxa+, head, 5dpf, GEX; Danio rerio; RNA-Seq
From scxa:mCherry zebrafish, we dissected and pooled 120 heads each for the 5 dpf scRNA-seq and multiome, 110 heads for the 5 dpf snATAC-seq, and 80 heads for the 14 dpf scRNA-seq experiment. Heads were partitioned into Eppendorf tubes on ice (12 heads per tube for 5 dpf and 8 heads per tube for 14 dpf) and washed twice in Ringer's solution, followed by enzymatic dissociation in pre-warmed 20 mg/mL Collagenase D (from stock of 400 mg/mL Collagenase D in HBSS), and 0.25% trypsin, 1 mM EDTA (pH 8.0) in PBS at 28.5°C for 1 to 1.5 h with vigorous pipetting every 10 min. The reaction was stopped with 6 mM CaCl2 and 30% fetal bovine serum (FBS) in PBS solution, and cells were collected by centrifugation at 2000 rpm for 5 min at 4°C. Cells from each stage were pooled into one tube after being resuspended and rinsed twice with 1% FBS, 0.8 mM CaCl2, 50 U/ml Penicillin, and 0.05 mg/ml Streptomycin in Leibovitz Medium solution and passed through a cell strainer. Cells were incubated with DAPI and subjected to fluorescence activated cell sorting (FACS). We collected approximately ~100,000 scxa:mCherry+ live cells (based on exclusion of DAPI) into a PBS solution with 0.04% BSA. Cells from each experiment were processed for nuclei isolation following 10X Genomics low cell count protocol (protocol CG000169) for snATAC-seq and Multiome, or used directly for scRNA-seq. Nuclei were imaged on a confocal microscope for absence of blebbing or abnormal morphology prior to proceeding. Cell and nucleus partitioning, barcoding, and library synthesis were performed following protocols of 10X Genomics for snMultiome (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression, protocol CG000338), scRNA-seq (Single Cell 3 ' Reagent Kits v2, protocol CG00052), and snATAC-seq (Chromium Next GEM Single Cell ATAC Reagent, protocol CG000209). Libraries were sequenced on the Illumina NextSeq/HiSeq platform to reach a depth of 60,000 and 45,800 reads per cell for 5 and 14 dpf RNA libraries, and 10,600 reads per nuclei for the 5 dpf ATAC library. The 5 dpf multiome had a depth of 9,000 reads per nuclei for the ATAC library and 736 genes per nuclei for the GEX library. Libraries were aligned against the reference genome built with the genomic sequence of GRCz11, the gene annotation of GRCz11.104, and the motif matrix of JASPAR2020; the mCherry sequence is built in as an extra chromosome.