GSM9015899: HCASMC, siCtrl, Rep4; Homo sapiens; RNA-Seq
The RNA was extracted from human coronary artery smooth muscle cells (hCASMCs) using the RNA extraction kit from Qiagen. Total RNA quality was determined by estimating the A260/A280 and A260/A230 ratios by nanodrop. RNA integrity was determined by running an Agilent Bioanalyzer gel, which measures the ratio of the ribosomal peaks. mRNA is purified from approximately 500 ng of total RNA with oligo-dT beads and sheared by incubation at 94°C. Following first-strand synthesis with random primers, second strand synthesis is performed with dUTP for generating strand-specific sequencing libraries. The cDNA library is then end-repaired, and A-tailed, adapters are ligated, and second-strand digestion is performed by Uracil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the Lab Chip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing. Samples are sequenced using 75 bp single end sequencing on an Illumina Hi-Seq 2500 according to Illumina protocols. Signal intensities are converted to individual base calls during a run using the system's Real Time Analysis (RTA) software.