GSM8735120: 3 μm_confine_rep3; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol Reagent (Invitrogen, 15596018). Total RNA was qualified and quantified as follows: (1) RNA purity and concentration were examined using NanoDrop 2000; (2) RNA integrity and quantity were measured using the Agilent 2100/4200 system. The 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 was synthesized using DNA polymerase I and dNTPs (dATP, dUTP, dCTP and dGTP). 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 without U was enriched by PCR, and the PCR product was purified to obtain the final library.