GSM7990936: Engineered heart issue_WT_3; Homo sapiens; RNA-Seq
For RNA extraction, tissues were flash frozen and crushed with plastic pestles. The aqueous phase was collected after TRIzol (Ambion) phase separation, and the RNA pellets were precipitated for total RNA extraction. Samples were treated with DNAse and cleaned with Qiagen RNeasy columns. Samples were sequenced by Yale Center for Genomic Analysis mRNA is purified from approximately 200ng of total RNA with oligo-dT beads and sheared by incubation at 94C in the presence of Mg (Roche Kapa mRNA Hyper Prep Cat# KR1352). Following first-strand synthesis with random primers, second strand synthesis and A-tailing are performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3' dTMP overhangs are ligated to library insert fragments. Library amplification amplifies fragments carrying the appropriate adapter sequences at both ends. Strands marked with dUTP are not amplified. Indexed libraries are quantified by qRT- PCR using a commercially available kit (Roch 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 are used for sequencing.