GSM6793305: TNFA_LO_3; Homo sapiens; RNA-Seq
Frozen tissue was processed using Biosafety Level 2+ (BL2+) procedures approved by the Beth Israel Deaconess Medical Center (BIDMC) Institutional Biosafety Committee (IBC). Brain tissues and primary neurons were homogenized using Trizol (Thermo Fisher) reagent and RNA extracted by phase separation. Total RNA was quantified by nanodrop (DeNovix DS-11) and TapeStation 4200 (RNA Screen Tape; Agilent Technologies, Inc). 450 ng of total RNA was used for library preparation via the KAPA RNA HyperPrep kit with RiboErase (HMR; Roche) according to the manufacturer's recommendations. Briefly, hybridization with hybridization oligos (HMR) was performed at 95oC for 2min followed by rRNA depletion using RNAse H which was performed at 45 oC for 30min. Following rRNA cleanup via KAPA pure beads, DNase digestion was performed at 37 oC for 30min followed by cleanup, RNA elution, fragmentation (6 min at 94oC for samples with RIN ≥ 7 or 5min at 85 oC for samples with RIN ≤ 7) and priming. First and second strand synthesis and A-tailing were performed according to manufacturer's recommendations. 1.5 uM KAPA Unique Dual-Indexed (UDI; Roche) adapters were ligated to the second strand synthesis product in the presence of a ligation master mix in a reaction that was performed at 20 oC for 15min. Following cleanup, all libraries underwent 10 cycles of amplification. Successful library production, quality control, and quantification was assessed using Tapestation (High sensitivity D1000 Screen Tape; Agilent Technologies, Inc). Libraries were pooled and sequenced via NovaSeq 6000.