GSM9131526: KO CTX; Mus musculus; RNA-Seq
Cells were prepared as described (doi:10.3791/65215) Cell number and viability were determined by a Trypan Blue exclusion assay on a Buerker Chamber. Samples were processed on the Chromium Controller from 10X Genomics (Leiden, The Netherlands). 16500 total cells were loaded per well into nanoliter-scale Gel Beads-in-Emulsion (GEMs). Single cell 3 prime mRNA seq library were generated using Chromium Next GEM Single Cell 3 prime Reagent Kits v3.1 (10X Genomics). Briefly, GEMs were generated by combining barcoded gel beads, a RT master mix containing cells, and partitioning oil onto Chromium Chip G. Following full length cDNA synthesis and barcoding from poly-adenylated RNAs, GEM were broken and pooled before cDNA amplification by PCR using 11 cycles. After enzymatic fragmentation and size selection, sequencing library were constructed by adding P5 and P7 primers (Illumina, San Diego, USA) as well as sample index via end-repair, A-tailing, adaptor ligation and PCR amplification with 12 cycles. Library quantification and quality control was performed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA).