GSM7401350: 18720X7 shCon-B; Mus musculus; RNA-Seq
Py230 single cell suspension was isolated by trypsinization and resuspended in phosphate buffered saline with 0.04% bovine serum albumin. The cell suspension was filtered through 40-micron cell strainer. Viability and cell count were assessed on Countess 2 (Invitrogen, Carlsbad, CA). Equilibrium to targeted cell recovery of 6,000 cells along with 10x Gel Beads and reverse transcription reagents were loaded to Chromium Single Cell Chip G (PN-1000120) to form Gel-Bead-In EMulsions (GEMs), the nano-droplets. Within individual GEMs, barcoded cDNA generated from captured mRNA was synthesized by reverse transcription at the setting of 53°C for 45 min followed by 85°C for 5 min. Subsequent fragmentation, end repair and A-tailing, adaptor ligation and sample indexing with dual index (PN-1000215) were performed in bulk according to the user guide.  The resulting barcoded libraries were qualified using Agilent D1000 ScreenTape on Agilent Technology 2200 TapeStation system and quantified by quantification PCR using KAPA Biosystems Library Quantification Kit for Illumine Platforms (KK4842). 10X Genomics Next GEM Single Cell 3' Gene Expression Library prep v3.1 with UDI