GSM9305206: pfc old saline 2; Mus musculus; RNA-Seq
Brain tissues were homogenized using Trizol (Thermo Fisher) reagent and RNA extracted by phase separation. 500ng of total RNA was used for library preparation via the KAPA RNA HyperPrep kit with RiboErase (HMR; Roche) according to the manufacturer's recommendations (Mavrikaki, Lee et al. 2022). Briefly, hybridization with hybridization oligos (HMR) was performed at 95 oC for 2 min. rRNA depletion using RNAse H was performed at 45 oC for 30 min. Following rRNA cleanup via KAPA pure beads, DNase digestion was performed at 37 oC for 30 min followed by cleanup, RNA elution, fragmentation (6 min at 94oC), 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 15 min. 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 together and subjected to NovaSeq 6000.