GSM7497686: WT_Sham rep2; Mus musculus; RNA-Seq
Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. 1 ug of total RNA was used for the construction of sequencing libraries. RNA libraries for RNA-seq were generated using Illumina's TruSeq RNA Library Prep Kit (Illumina, San Diego, CA) following the manufacturer's protocols. Briefly, mRNA is enriched by using the oligo (dT) magnetic beads. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The products were then purified and amplified using PCR.