GSM8829187: MEFs +Myf6, Day 2, lenti, rep2; Mus musculus; RNA-Seq
To extract total RNA from cells in culture, cells were trypsinized for 5 minutes at 37°C, washed 1x with PBS and the RNA isolated using the RNeasy Mini Kit, including DNAse digestion. Samples were stored at -80°C until library preparation. Library preparation from RNA was performed either at the Functional Genomics Center Zurich (FGCZ) of University of Zurich and ETH Zürich (Myf6-induced lentiviral reprogramming) or at Novogene UK (TetO-Myf6 and MyoD-KO reprogramming). At the FGCZ, the RNA quality was determined with a Fragment Analyzer (Agilent Technologies) and only samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq Stranded mRNA (Illumina) was used for library preparation. Briefly, total RNA samples (100-1000 ng) were polyA enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and adenylated before ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR.  The quality and quantity of the enriched libraries were validated using the Fragment Analyzer (Agilent Technologies): the product was a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20 before sequencing. At Novogene, RNA quality was measured with the 5400 Fragment Analyser (Agilent Technologies) and only samples with RNA Integrity Number (RIN)≥ 9.1 were used for library preparation. Library preparation was carried out with a modified kit based on the TruSeq RNA Library Prep Kit (Illumina). Briefly, mRNA was purified from the total RNA samples by polyA enrichment using polyT oligo-attached magnetic beads. RNA was then fragmented and the first cDNA strand synthesized using random hexamer primers. The second strand of cDNA was then synthesized using dTTP (non-directional library), end-repaired, A-tailed, ligated with adapters, size selected, digested with USER enzyme, amplified and purified. The library concentration was checked with Qubit Fluorometer and RT-qPCR, while library fragment size was checked on a bioanalyzer.