GSM3770855: RNA-seq-CRISPRk-12042-GH550_1; Homo sapiens; RNA-Seq
2e7 K562 cells per replicate were pelleted at 500 g for 5 minutes at 4°C and then resuspended in 1x PBS buffer. Two replicates were performed for each sgRNA. RNA extraction was performed as follows: 500 uL of TRIzol was added to each sample, mixed by tube inversion, and then 5 minutes later 100 uL of chloroform was added. Samples were spun at 12,000 g for 15 minutes at 4°C. The aqueous layer was transferred to an RNase-free tube and mixed with 300 uL of 70% ethanol and vortexed. Contents were then transferred to Direct-zol miniprep columns (Zymo) and the protocol was followed according to the manufacturer's instructions, including the on-column DNaseI treatment. RNA was eluted in 15 uL of RNase-free water and stored at -80°C and a separate 2 uL aliquot was set aside for testing RNA concentration and quality via Nanodrop. RNA-seq libraries were prepared from 700 ng of total RNA with the TruSeq RNA Library Prep kit v2 (Illumina), which uses oligo-dT beads to enrich for A-tailed mRNAs.