GSM5353221: PA_PB2A_Pool_1_3_S25_L001; Homo sapiens; RNA-Seq
Tissue samples were minced with surgical scissors and washed with RP-10 (RPMI + 10% FBS). Each sample was centrifuged at 1200 rpm for 5 minutes, resuspended in 10 mL digestive media (HBSS + 1% HEPES) with Liberase TM (Roche, Cat: 5401119001) or 1000 U/mL collagenase type IV (Worthington, Cat: LS004188), and rotated for 30 minutes at 37 °C. Samples were triturated by pipetting ten times after every 10 minutes during the incubation or by pipetting 15 times at the end of the incubation. Each sample was filtered through a 70 µm filter (Falcon, Cat: 352350), washed with RP-10, centrifuged at 1200 rpm for 5 minutes, washed again with RP-10, and resuspended in RP-10. A hemocytometer was used to count the cells. Libraries were prepared for each pool, using 800-1000 pg of DNA and the Nextera XT DNA Library Preparation Kit. They were dual-indexed with N700 and N500 oligonucleotides. Library products were purified using SPRI beads, first at 0.6x and then at a 1x volumetric ratio. Libraries were then run on an HSD1000 tape (Agilent, Cat: 50675584) to determine the concentration between 100-1000 bp. For each library, 3 nM dilutions were prepared.