GSM2774067: PGC-1α-KD C2C12 + FLAG-PGC-1α(ΔCBM) 2; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol (Ambion). The TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) was used for next-generation sequencing library construction following the manufacturer's protocols. Briefly, poly(A)+ RNA was purified from 200 ng of total C2C12-cell RNA using oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed using random hexamer priming followed by second-strand cDNA synthesis using dUTP incorporation for strand marking. The resulting double-stranded cDNA was then subjected to end repair and 3´-end adenylation. Illumina adaptors were ligated to both cDNA ends. Adapted cDNA was purified using Ampure beads and PCR-amplified using primers specific to the adaptor sequences to generate cDNA amplicons of approximately 200-500 base-pairs. Amplified libraries were hybridized to the Illumina single-end flow cell and further amplified using the cBot (Illumina, San Diego, CA). Single-end reads of 100 nucleotides were generated for each sample using HiSeq2500v4 (Illumina).