title: GSM4832650: CD42 positive megakaryocytes RNA-seq replicate 2; Homo sapiens; RNA-Seq
library_construction_protocol: Cell lysis and RNA isolation for RNA sequencing was performed according to Ignolia et al. with some adaptations. Briefly, CD42- and CD42+ selected megakaryocytes were pelleted. Cell pellets were dissolved in 150uL ice cold lysis buffer (20mM Tris pH 7.4, 250mM NaCl2, 5mM MgCl2, 0.5% Triton X-100, 1mM Dithiotreitol (DTT), 0.024U/uL Turbo DNAse (Ambion), 0.01U/uL RNAseOUT (Invitrogen) and 100ug/mL Cycloheximide). The lysate was immediately split in two aliquots of 50uL for total RNA-sequencing. 2.5uL SuperaseIN RNAse inhibitor (Invitrogen) was immediately added to the total RNA sample, and total RNA was extracted using the miRNAeasy kit (Qiagen) according to manufacturer's protocol. RNA quality was assessed on Agilent bioanalyzer RNA chip (all samples with RIN >9.0), and quantified using Qubit 2.0 (Thermofisher). Ribosomal RNA was removed using Ribo Zero Gold (Epicentre/Illumina; 20020596) using the manufacturer's protocol, and the cDNA library was constructed with the KAPA stranded RNA-seq kit (KAPA biosystems KK8400) according to manufacturer's protocol with Illumina Truseq forked adapters. Library quality was assessed on Agilent Bioanalyzer High sensitivity DNA chip and quantified using qPCR with the KAPA library quantification kit (KK4824). Libraries were pooled to a 2nM concentration and 125bp paired-end reads were sequenced on HiSeq 2000 (Illumina).