GSM6383049: VWAT_HFD_4; Mus musculus; RNA-Seq
RNA was isolated using Direct-zol RNA Miniprep Kit (Zymo Research). mRNA was purified from approximately 200ng of total RNA with oligo-dT beads and sheared by incubation at 94C in the presence of Mg2+  (Roche Kapa mRNA Hyper Prep Cat# KR1352).  Following first-strand synthesis with random primers, second strand synthesis and A-tailing was performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3' dTMP overhangs were ligated to library insert fragments.  Library amplification amplified fragments carrying the appropriate adapter sequences at both ends.  Strands marked with dUTP were not amplified. Indexed libraries were quantified by qRT-PCR using a commercially available kit (Roche KAPA Biosystems Cat# KK4854) and insert size distribution determined by either the Agilent Bioanalyzer. Samples with a yield of ≥ 0.5 ng/ul and a size distribution of 150-300bp were used for sequencing.  For flow cell preparation and sequencing, sample concentrations were normalized to 15 pM and loaded onto an Illumina HiSeq 2500 V4 flow cell at a concentration that yields 25 million passing filter clusters per sample. Samples were sequenced using 75 bp single-end sequencing according to Illumina protocols. The 6 bp index is read during additional sequencing reads that automatically follow the completion of read 1.  Data generated during sequencing runs are simultaneously transferred to the YCGA high-performance computing cluster. A positive control (prepared bacteriophage Phi X library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time. Signal intensities were converted to individual base calls during a run using the system's Real Time Analysis (RTA) software.  Base calls were transferred from the machine's dedicated personal computer to the Yale High Performance Computing cluster via a 1 Gigabit network mount for downstream analysis.  Primary analysis - sample de-multiplexing and alignment to the mouse genome - was performed using Illumina's CASAVA 1.8.2 software suite. Only samples with an error rate less than 2 % and with a distribution of reads per sample in a lane within reasonable tolerance were further processed.