GSM8864249: WT3-exp3; Homo sapiens; RNA-Seq
Total RNA was extracted using a Qiagen Rneasy micro kit, according to manufacturer's recommendations. CATseq (RNA-seq on CRISPR cells) collection and analysis: RNA-seq libraries were prepared using CATS mRNA-seq kit v2 (with poly(A) selection) with dual indexing (Diagenode, Belgium) as per the manufacturer's protocol. Briefly, 250 ng of total RNA was spiked-in with SIRV Set 3 (Lexogen GmbH, Austria) calculated based on 1% mRNA and 1% spike-in as per the manufacturer's protocol was used to generate cDNA. The resulting libraries were quantified with Qubit DNA High Sensitivity assay (ThermoFisher). Fragment sizes were analyzed on the Agilent Bioanalyzer using the High Sensitivity DNA assay prior to sequencing. Paired-end 50-bp sequencing was performed at The Centre for Applied Genomics (TCAG, Canada) on an Illumina HiSeq2500 Rapid Run (Illumina, USA) with cycles extended to 68 bp using a custom paired-end sequencing primer for read 2 (Diagenode) and the following dark cycle recipe 5DC+58(R1)+8(Index1)+8(Index2)+5DC+58(R2).