title: GSM3872552: 2161-4_quad_ex_WT_of_MMVVg; Mus musculus; RNA-Seq
library_construction_protocol: To extract high quality total RNA, the quadriceps muscles were processed first in the Trizol reagent (ThermoFisher Scientific, Catalog # 15596018) and then purified by RNeasy mini columns (Qiagen, catalog # 74104) as follows. To avoid sampling bias, the entire quadriceps muscle were homogenized in Trizol reagent at the weight (mg) to volume (µL) ratio of 1:15. One mL of homogenate was processed following the manufacture's protocol until the step of phase separation. Then, 0.5 mL of the aqueous phase was mixed with an equal volume of 70% ethanol for subsequent RNA purification by RNeasy mini kit (Qiagen, catalog # 74104) with on-column DNAse (RNase-Free DNase Set, Qiagen catalog # 79254) treatment. The concentration of the RNA was determined by Qubit fluorometric quantitation (Qubit RNA BR Assay Kit, ThermoFisher Scientific, catalog # Q10210). The integrity of the total RNA was determined by a Fragment Analyzer (Advanced Analytical Technologies, Inc) to be greater than 9.0. 1 µg of total RNA from each sample was used for RNA sequencing library preparation with the TruSeq® Stranded mRNA Library Prep kit (Illumina, catalog # 20020594). The libraries were barcoded by TruSeq® RNA Unique Dual Indexes (Illumina, catalog # 20022371) and quantified by qPCR on a Bio-Rad CFX Connect Real-Time PCR detection system with the NEBNext Library Quant Kit for Illumina (New England Biolabs, catalog # E7630L). The libraries were normalized to 10 nM, pooled and sequenced. RNA sequencing was carried out at Johns Hopkins School of Medicine Genetic Resources Core Facility on a NovaSeq 6000 sequencing system (Illumina) with a S1 flow cell for 200 cycles, generating 100 bp paired-end reads.