GSM5021119: Gemini-72-treated (1 week); Mus musculus; RNA-Seq
Mice prostates were mechanically and enzymatically dissociated. Single living cells were obtained by FACS sorting. Cell number and viability were determined by a Trypan Blue exclusion assay on a Neubauer Chamber. Samples consisted of  > 95% viable cells were processed on the Chromium Controller from 10X Genomics (Leiden, The Netherlands). Ten thousand cells were loaded per well to yield approximately 5 to 6,000 captured cells into nanoliter-scale Gel Beads-in-Emulsion (GEMs). Single cell 3' mRNA seq library were generated according to Chromium Single Cell 3' Reagent Kits User Guide (v2 Chemistry) from 10X Genomics (ref CG00052 Rev E). Briefly, GEMs were generated by combining barcoded gel beads, a RT master mix containing cells, and partitioning oil onto Chromium Chip A. Following full length cDNA synthesis and barcoding from poly-adenylated mRNA, GEM were broken and pooled before cDNA amplification by PCR using 10 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 12 cycles. Library quantification and quality control was performed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA).