title: GSM6048483: RENCA Parental rep 2; Mus musculus; RNA-Seq
library_construction_protocol: RNA from Renca Parental, Sdhb-KO and Fh1-KO was isolated using RNeasy microcolumns (Qiagen) and quantified using NanoDrop 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.