title: GSM9161587: Human Tconv stim, Donor 1, CRISPRoff AAVS1, gRNA gJ-48; Homo sapiens; RNA-Seq
library_construction_protocol: To isolate RNA, cells were pelleted and resuspended in 300 µL RNA Lysis Buffer (Zymo Research, catalog no. R1060-1-50), vortexed vigorously, and frozen at -80°C until further processing. Lysed cells were thawed, and RNA was isolated using the QuickRNA Microprep Kit (Zymo Research, catalog no. R1050), according to the manufacturer's protocol, but skipping the DNase I treatment. Eluted RNA in 15 µL was treated with 1.5 µL TURBO DNase in 1X TURBO DNase Buffer (Invitrogen, catalog no. AM2238) and incubated 20 minutes at 37°C. RNA samples were cleaned up using the RNA Clean & Concentrator-5 Kit (Zymo Research, catalog no. R1013), according to the manufacturer's protocol. RNA integrity (RIN score > 8.0) and yield was assessed using Qubit RNA HS Assay Kit (Fisher Scientific, catalog no. Q32855) and Tapestation High Sensitivity RNA ScreenTape Analysis (Agilent, catalog nos. 5067-5579 and 5067-5580). Libraries for RNA sequencing were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche, catalog no. 08098131702) and KAPA Unique Dual-Indexed Adapter Kit (Roche, catalog no. 08861919702), according to manufacturer's protocol.