title: GSM7030508: TB6545, RSL3; Homo sapiens; RNA-Seq
library_construction_protocol: Tissue slices were collected for dissociation using Papain Dissociation System (Worthington) as previously described(Mizrak et al., 2020; Mizrak et al., 2019) with following modifications. Briefly, minced tissue was digested with papain (Worthington, 10 units per sample) in PIPES solution (120 mM NaCl, 5 mM KCl, 20 mM PIPES (Sigma), 0.45% glucose, DNase I (Worthington, 100 units per sample), 1xAntibiotic/Antimycotic (GIBCO), pH adjusted to 7.6) for 30 min at 37°C on gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec, program 37C_ABDK). After digestion, the cell suspension was applied to a MACS SmartStrainer (70 μm, Miltenyi Biotec) and centrifuged at 300xg for 10 minutes at 4°C. The cell pellets were then subjected to red blood cell removal (Red Blood Cell Lysis Solution (10x), Miltenyi Biotec) and debris removal (Debris Removal Solution, Miltenyi Biotec) according to the manufacturer's instructions. mRNA molecues were captured using bead-bound oligo dT(30). Captured mRNA molecues were reverse transcriped to obtain cDNA which is then amplified by PCR reactions. The cDNA PCR reaction product was then purified with AMPure XP beads. The purified cDNA PCR product was used as the input for Nextera XT reactions to generate sequencing library.