GSM5628770: Control, replicate 1; Mus musculus; RNA-Seq
Total RNA was extracted by Direct-zol RNA extraction Kit by Zymo Research. mRNA (polyA) purification, RNA fragmentation, first and second strand cDNA synthesis were performed according to the TruSeq RNA Sample Preparation v2 Kit using Superscript III for reverse transcription (50-min incubation at 50 degrees celsius). cDNA was purified with AMPure XP beads diluted 1:2 with 20% PEG, 1.25M NaCl, and eluted in 27.5 μl of 10 mM Tris-HCl pH 7.9, 25 μl of which were transferred to a new tube and subjected to library construction. RNA-seq libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB E7645), diluting NEB adaptors 1:5 and performing 8 PCR cycles.