GSM5585125: chd8_WT; Danio rerio; RNA-Seq
chd8+/+ and homozygous chd8 sa19827/sa19827 male adult zebrafish were euthanized in 800 µg/ml tricaine solution. The fish were dissected, their guts were harvested and placed in RPMI at room temperature. The guts were rolled on paper moistened with RPMI to remove the fat residue, then placed in RPMI with 10% fetal calf serum and cut into small pieces that were placed in 1 ml of digestion medium (1 ml of RPMI - 12 µl of activated fetal calf serum - 10 mg of dispase collagenase) for 15 minutes, at 37 °C, under agitation at 500 rpm. The cells were then filtered on a cell filter (diameter 40 µm, Dutscher, ref 141378C), using the plunger of 1 ml syringe. Cell number and viability were determined by a Trypan Blue exclusion assay on a Neubauer Chamber. Samples consisted of > 90 % viable cells were processed on the Chromium Controller from 10X Genomics (Leiden, The Netherlands). 10,000 total cells were loaded per well to yield approximately 6,500 captured cells into nanoliter-scale Gel Beads-in-Emulsion (GEMs). Single cell 3' mRNA seq library were generated according to 10X Genomics User Guide for Chromium Single Cell 3' Reagent Kits (v3 Chemistry). Briefly, GEMs were generated by combining barcoded gel beads, a RT master mix containing cells, and partitioning oil onto Chromium Chip B. Following full length cDNA synthesis and barcoding from poly-adenylated mRNA, GEMs were broken and pooled before cDNA amplification by PCR using 11 cycles. After enzymatic fragmentation and size selection, sequencing library were constructed by adding Illumina (Paris, France) P5 and P7 primers as well as sample index via end repair, A tailing, adaptor ligation and PCR with 14 cycles. Library quantification and quality control was performed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA).