GSM7430894: E14_MES cells, WT, Rep 2; Mus musculus; RNA-Seq
For RNA-seq, 1 × 106 MES or CP cells were harvested and RNA extraction was performed using Rneasy mini plus kit (Qiagen). 1 ?g of total RNA was used for the construction of sequencing libraries and sequencing.RNA library for RNA-seq was prepared as follows: mRNA was purified from total RNA using polyT and then fragmented into 300~350 bp fragments, the first strand cDNA was reverse-transcribed using fragmented RNA and dNTPs (dATP, dTTP, dCTP and dGTP) and second strand cDNA synthesis was subsequently performed. Remaining overhangs of double-strand cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3? ends of DNA fragments, sequencing adaptors were ligated to the cDNA and the library fragments were purified. The template was enriched by PCR, and the PCR product was purified to obtain the final library. RNA library for RNA-seq was prepared as follows: mRNA was purified from total RNA using polyT and then fragmented into 300~350 bp fragments, the first strand cDNA was reverse-transcribed using fragmented RNA and dNTPs (dATP, dTTP, dCTP and dGTP) and second strand cDNA synthesis was subsequently performed. Remaining overhangs of double-strand cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3? ends of DNA fragments, sequencing adaptors were ligated to the cDNA and the library fragments were purified. The template was enriched by PCR, and the PCR product was purified to obtain the final library.