GSM5856925: 210acKO_Early_Rep1; Mus musculus; RNA-Seq
Hearts were removed, flash frozen in liquid nitrogen, and RNA was harvested using Trizol reagent. The RNA was treated by DNase I followed by Pheno-Chroloform extraction. The total RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer's protocols. Briefly, mRNA was purified from 100 ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3` adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100-nt are generated for each sample and aligned to the organism specific reference genome.