GSM4279265: siRBM26.A: RNA-Seq of HAP1 cells after knockdown of RBM26, replicate 1; Homo sapiens; RNA-Seq
Total RNA was extracted with the Qiagen RNasy Mini kit. DNase-treated RNA samples were submitted for RNA-seq at the Donnelly Sequencing Centre at the University of Toronto (http://ccbr.utoronto.ca/donnelly-sequencing-centre). Total RNA was quantified using Qubit RNA BR (cat # Q10211, Thermo Fisher Scientific Inc., Waltham, USA) fluorescent chemistry and 1 ng was used to obtain RNA Integrity Number (RIN) using the Bioanalyzer RNA 6000 Pico kit (cat # 5067-1513, Agilent Technologies Inc., Santa Clara, USA). The lowest RIN was 8.7; median RIN was 9.6.  2.5 ug of total RNA per sample was processed using the MGIEasy Directional RNA Library Prep Set V2.0 (protocol v. A0, cat # 1000006385, Shenzhen MGI Tech Co. Ltd., Shenzhen, China) including mRNA enrichment with Dynabeads mRNA Purification Kit (cat # 61006, Thermo Fisher Scientific Inc.). RNA was fragmented at 87Â°C for 6 minutes, after adding only 75% of the recommended volume of fragmentation buffer in order to produce longer fragments. Libraries were amplified with 12 cycles of PCR.  1 ul top stock of each purified final library was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip (cat # 5067-4626, Agilent Technologies Inc., Santa Clara, USA) to determine an average library size of 581 bp, and to confirm absence of dimers. The libraries were quantified using the Quant-iT dsDNA High Sensitivity (cat # Q33120, Thermo Fisher Scientific Inc., Waltham, USA) fluorometry kit, pooled equimolar, and libraries in each of 4 replicate pools were then circularized using the MGIEasy Circularization Module (cat # 1000005260, Shenzhen MGI Tech Co. Ltd.).  From each of the 4 pools, 40 fmol of circularized library was sequenced 2x150 bp on a single lane of an FCL flowcell on the MGISEQ-2000 platform (also known as DNBSEQ-G400 platform, Shenzhen MGI Tech Co. Ltd.), for a total of 4 lanes of sequencing.