GSM4730101: WT2_tail_tip_fibroblasts; Mus musculus; RNA-Seq
Total RNA was extracted from cells grown on a 15cm tissue culture plate using Trizol (ThermoFisher), followed by DNase treatment using TURBO DNA-freeTM Kit (ThermoFisher). Polyadenylated mRNA was isolated from total RNA using DynabeadsTM mRNA Purification Kit (ThermoFisher). RNA integrity was checked by Bioanalyzer (Agilent 2100), followed by fragmentation with Mg2+- catalyzed hydrolysis. cDNA library was constructed as previously described (Mortazavi et al. 2008). In brief, the first-strand cDNA was reverse transcribed with random primers, followed by dA addition and adaptor ligation. Then an RNA gel-purified was performed for size selection of 300 bp, follow by UNG treatment for strand specificity and PCR amplification. cDNA sequencing was performed as previously reported (Mortazavi et al. 2008). DNA quality and size were checked by Bioanalyzer (Agilent 2100), quantified by KAPA Library quantification Kit (Illumina), followed by sequencing on NextSeq500 (Illumina) using paired-end mode.