GSM5651437: MNGC245: PtenL2/L2; 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 were processed on the Chromium Controller from 10X Genomics (Leiden, The Netherlands). Ten thousand cells were loaded per well. Single cell 3' mRNA seq library were generated according to Chromium Single Cell 3' Reagent Kits User Guide (v2 Chemistry [Longitudinal study of luminal cells] / v3 Chemistry [Pten(i)pe-/-, PtenL2/L2 and Pten/Hif1a(i)pe-/-] ) from 10X Genomics (ref CG00052 Rev E [Longitudinal study of luminal cells] / CG000183 Rev A [Pten(i)pe-/-, PtenL2/L2 and Pten/Hif1a(i)pe-/-]). Briefly, Gel Beads-in-Emulsion (GEMs) were generated by combining barcoded gel beads, a RT master mix containing cells, and partitioning oil onto Chromium Chip. Following full length cDNA synthesis and barcoding from poly-adenylated mRNA, GEM were broken and pooled before cDNA amplification by PCR. 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. Library quantification and quality control was performed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA).