GSM5175871: D4_MyoD_1; Mus musculus; RNA-Seq
Cultured cells were harvested using trypsin and total RNA was extracted using RNeasy mini kit (Qiagen, 74104). FACS-purified cells were directly sorted into cell lysis buffer and processed for total RNA extraction using the same kit. The RNA quality was measured with a Fragment Analyzer (Agilent, Santa Clara, California, USA) and only those samples with a 28S/18S ratio over 1.9 were used for library construction according to the TruSeq Stranded mRNA protocol (Illumina, Inc., California, USA). Briefly, 100-1000 ng of ploy A-enriched total RNA was reverse-transcribed into double-stranded cDNA. The cDNA was fragmented, end-repaired and adenylated. Then, TruSeq adapters containing unique dual indices (UDI) were ligated to processed cDNA for multiplexing. Using PCR, fragments containing TruSeq adapters on both ends were selectively enriched and the quality and quantity of the enriched libraries were determined using a Fragment Analyzer, which showed an average fragment size of approximately 360 bp. As a final step, the libraries were diluted in a concentration of 10 nM in Tris-Cl buffer (10 mM, pH8.5) with 0.1% Tween 20 and used for sequencing. The sequencing was carried out on Novaseq 6000 (Illumina, Inc., California, USA) with 100bp single end reads according to standard protocol.