GSM2776390: UC338; Homo sapiens; RNA-Seq
Snap frozen muscle biopsies were sectioned with a Thermo Scientific Microm HM 560 cryostat (Thermo Scientific, Wilmington, DE). Fifteen 10 μm cryosections were collected in Trizol (Life Technologies, Carlsbad, CA). The RNA was isolated using standard Trizol protocol, and further purified using miRNeasy Mini Kit (Qiagen, Valencia, CA). After isolation, the RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). The RNA samples were analyzed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) to obtain RNA integrity numbers (RIN) as measure of quality. Only samples having a RIN quality ≥ 6 were subject to RNA-Seq Analysis. Libraries were prepared from isolated RNA according to Illumina protocols (Illumina, Inc, San Diego, CA) and sequenced by Beckman Coulter Genomics (Danvers, Massachusetts). Briefly, mRNA libraries were constructed from 150 ng of total RNA using the Illumina TruSeq RNA Sample Prep Kits, version 2. The resulting cDNA was fragmented using a Covaris E210. Library amplification was performed using 12-15 cycles to minimize the risk of over-amplification. Unique barcode adapters were applied to each library. Equal volumes of individual libraries were pooled and run on a MiSeq. The libraries were then repooled based on the MiSeq demultiplexing results and sequenced on a HiSeq 2000 with version 3 flow cells and sequencing reagents. On average, 69 million 100-base read pairs were generated for each individual library. Data was processed using RTA 1.13.48 and deplexed via CASAVA 1.8.2.