GSM7989104: Faki-treated_L002; Mus musculus; RNA-Seq
Single cells were stained using trypan blue for visualization, counting and viability assessment using the Countess II Automated Cell Counter and diluted as needed to a final concentration of 700-1200 cells/ml in 1x PBS + 0.04% BSA. Single cells were stained using trypan blue for visualization, counting and viability assessment using the Countess II Automated Cell Counter and diluted as needed to a final concentration of 700-1200 cells/ml in 1x PBS + 0.04% BSA.  A minimum of two reproducible counts with SD <25% were required before droplet preparation. Cell viability was 75-76%. A total of 8832 for the vehicle- and 8437 cells for the FAKi-treated samples were used for sequencing. Cells were processed with Chromium Next Gem single Cell 3' GEM Library and Gel Bead Kit v 3.1 (10X Genomics) according to manufacturer instructions. Briefly, cells diluted in master mix containing reverse transcription reagents and primer and were transferred to the ChromiumChip with gel beads and partitioning oil for preparation of nanoliter-scale Gel Bead-In-Emulsions (GEMs). Reverse transcription produced cDNA with a cellular 10X barcode and UMI, and was recovered with Dynabeads MyOne SILANE magnetic beads.  Subsequently the cDNA underwent 12 cycles of amplification before clean-up using SPRIselect Reagent (Beckman).  Total cDNA yield was calculated following quality assessment (High Sensitivity D5000 ScreenTape, Agilent) and fluorometric quantitation (Qubit, ThermoFisher). Libraries were constructed by enzymatic fragmentation, end repair and A-tailing according to the manufacturer's instructions.  A double sided SPRIselect Reagent (Beckman) clean up prepared samples for adapter ligation and another round of magnetic bead clean-up before sample indexing PCR for 12 cycles. A further double sided size selection produced libraries of approximately 400bp, which were quantified for paired end sequencing (26bp x 98bp).