GSM8880143: MPC cells, control rep2; Mus musculus; RNA-Seq
The concentration, quality and integrity of total RNA were determined using a NanoDrop spectrophotometer (Thermo Scientific). 3μg of RNA were used as input material for the RNA sequencing. Firstly, mRNA was purified by using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First and second strand cDNA was synthesized using random oligonucleotides with Super Script II and DNA Polymerase I with RNase H, respectively. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent).