title: GSM5393633: C2C12 cell line - siCTRL_rep1; Mus musculus; RNA-Seq
library_construction_protocol: The total RNA of C2C12 cells treated with siRNA, plasmid and virus was extracted with Trizol reagent (#15596018, Invitrogen). For cDNA library preparation, total RNA was isolated using Trizol from C2C12 cells treated with Control (n=3) or Cox7a2l (n=3) siRNA. RNA quality was determined with agarose gel and optical density. Only samples with a 260 nm/280 nm ratio within 1.8-2.1 and the intensity of a 28S/18S ratio between 1.5-2 were further processed. The cDNA library was prepared as previously described (Zhang et al., 2012). Briefly, total RNA sample (2 ug) were performed rRNA depletion and RNA fragmentation. Then the RNA was reversetranscribed into double-stranded cDNA with random primer. cDNA samples were end-repaired and polyadenylated before ligation of Y-shaped adapter containing the index for multiplexing, then performed the dUTP strand degradation. Fragments containing Y-shaped adapter on both ends were  enriched by PCR. The quantity of the cDNA library was validated using Qubit dsDNA HS Assay Kit (#Q32851, Invitrogen). The purity of the libraries was analyzed by fragment analyzer automated CE system (Advanced Analytical) and the sequencing was further performed only when the libraries had a size of 150 bp to 300 bp and no adaptor dimer contanmination. Sequencing was performed on the illumina novaseq 6000.