GSM5283742: EZH1 HET rep2; Danio rerio; RNA-Seq
Pools of fluorescent zebrafish embryos/larvae were dissociated using Liberase TM (Sigma) in a 34C circulating water bath and resuspended in 1X PBS/1mM EDTA. kdrl:GFP+ cells were FACS sorted on a FACSAria II (BD) at the BCH Flow Cytometry Core into 1X PBS containing 2% fetal bovine serum. Single-cell RNA-seq was performed on the 10X Genomics Chromium platform using the single cell expression 3' v2 profiling chemistry. Following kdrl:GFP+ FACS, cells from each ezh1 genotype were loaded across two lanes in a 10X Genomics Single Cell 3' Chip as independent technical replicates and libraries were constructed according to the manufacturer's protocol. Cells were loaded into 10X lanes at cell concentrations to maintain an estimated doublet rate below 5%. The final libraries were assayed via an Agilent High Sensitivity dsDNA Bioanalyzer, normalized, pooled and shallow sequenced (Illumina MiniSeq), identifying ~11,000 high confidence cell barcodes in total across all libraries. The libraries were subsequently renormalized per the distribution of reads/library from the shallow sequencing run and deep sequenced on a NovaSeq S4 (Illumina) to a depth of ~100,000 reads per cell with cycle configuration 151-8-8-151.