GSM9288619: CC, day7, graft4; Homo sapiens; RNA-Seq
RNA was isolated from 30 mg pieces of each graft using the Qiagen AllPrep DNA/RNA kit and quality was confirmed by measuring RNA concentration and absorbance ratios A260/A230 and A260/A280 using the NanoDrop spectrophotometer. 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.