title: GSM6426677: e13.5heart_Nos1ap_null_male_rep1; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from e13.5 heart tissue using RNeasy Mini Kit following manufacturers' recommendations (Qiagen, MD) that included the on-column DNase digestion using RNase-Free DNase set (Qiagen, MD). KAPA Stranded mRNA-Seq kit (KAPA Biosystems, MA) was used to generate indexed Illumina platform sequencing libraries.  Briefly, polyA RNA was captured from 1µg total RNA using magnetic oligo-dT beads.  After elution from the magnetic beads, polyA RNA was fragmented to generate inserts ranging in size from 100-200 bp, followed by random priming and reverse transcription to generate double-stranded cDNA.  Next, after performing a 1.8× SPRI cleanup using AMPure XP beads (Agencourt, IN), dAMP was added to 3'-ends of the cDNA fragments. Indexed 3'-dTMP Illumina TruSeq adapters were ligated to cDNA fragments.  Ligated fragments were subsequently size selected using PEG/NaCl SPRI solution and underwent PCR amplification (12 cycles) to generate the sequencing libraries.  After performing a 1× SPRI cleanup using AMPure XP beads (Agencourt, IN), a sample from each library was used to assess library fragment size distribution by electrophoresis using BioAnalyzer High Sensitivity DNA Assay (Agilent Technologies, CA) and to assess library concentration by qPCR using KAPA library quantification kit (KAPA Biosystems, MA).  Equimolar amounts of libraries were pooled and sequenced on an Illumina HiSeq 2500 instrument using standard protocols for paired end 100 bp sequencing with a desired sequencing depth of ~30 million paired end reads per library.