GSM5235367: Immortalized Human Bone Marrow Mesenchymal Stromal Cells - hTERT (iMSC3), A1; Homo sapiens; RNA-Seq
Total RNAs were extracted from all groups A, B, C and D. The harvested cells were first lysed using Qiazol lysis reagent. Then, the extraction was carried out using miRNeasy extraction kit following the manufacturer's instructions (Qiagen, Germany). The RNA quantity and purity were determined using Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments, preferentially\preferably  of 150-200  bp in length, the library fragments were purified using AMPure XP system (Beckman Coulter, USA). Then, 3µl of USER Enzyme (NEB, USA) was used with size-selected, adaptor ligated cDNA at 37°C for 15 min followed by 5 min at 95°C. After that, PCR was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, the PCR products were purified (AMPure XP system) and library quality was assessed using the Agilent Bioanalyzer 2100 system.