GSM4820208: MSCORS donor 30 passage 2; Homo sapiens; RNA-Seq
Cultivation medium was removed, and cells were rinsed with PBS and harvested using QiazolTM (Qiagen, DE) reagent and isopropanol precipitation. RNA purity was measured using NanoDrop™One/OneC for ratios of OD260/280 and OD260/230. RNA was quantified using Qubit™RNA HS Assay Kit on Invitrogen Qubit® 3.0 Fluorometer. The integrity of RNA (RNA Integrity Number, RIN) was determined using Agilent 4200 TapeStation system 1 μg of total RNA for the construction of sequencing libraries. Poly-A-tailed mRNA was captured from the total RNA sample using oligo dT magnetic beads Dynabeads® Oligo(dT)25 (ThermoFisher, DE). As RNA template, selected RNA was heat fragmented, randomly primed using random primers (Invitrogen, CA) as RNA template, and complementary single-stranded cDNA was synthesized by M-MuLV (Biolabs, UK) reverse transcriptase. mRNA was degraded using RNaseH (2U/ml), and second strand of cDNA was synthesized by DNA polymerase I using dNTPs. Purified double strand cDNA was end-repaired using End Repair Buffer (Enzymatics, MA), and dA-tailed using DNA polymerase Klenow Fragment (3→5´exo-, Biolabs, UK) and dATP, adapter ligated of indexed adapters. cDNA of 200bp size was selected using AMPure XP beads (Beckman Coulter, US), and amplified via PCR. After purification, dscDNA library was quantified using Kapa qPCR, and the size of cDNA fragments was determined by Agilent 4200 TapeStation. After equimolar pooling, the samples were sequenced using Illumina PE150 kits.