GSM8491027: D3_Teff_Stimulated_MED14_KO_RNA; Homo sapiens; RNA-Seq
8 and 10 days after editing, respectively, resting and 48-hour restimulated cells were pelleted and resuspended at 1x106 cells per 300 µL of RNA lysis buffer (Zymo, #R1060-1-100). Cells were pipette mixed and vortexed to lyse and frozen at -80 until RNA isolation was performed. RNA was isolated using the Zymo-Quick RNA micro prep kit (#R1051) according to the manufacturer's protocol with the following modifications: After thawing the samples, each sample was vortexed vigorously to ensure total lysis prior to loading into the extraction columns. The optional kit provided DNAse step was skipped, and instead RNA was eluted from the isolation column after the recommended washes and digested with Turbo-DNAse (Fisher Scientific, AM2238) at 37 C for 20 minutes. Following digestion, RNA was purified using the RNA Clean & Concentrator-5 kit (Zymo, #R1016) according to the manufacturer's protocol. The purified RNA was submitted to the UC Davis DNA Technologies and Expression Analysis Core to generate 3′ Tag-seq libraries with unique molecular indices (UMIs). Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen) for multiplexed sequencing.