title: GSM7074103: BM5; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was purified with Qiagen Rneasy PLUS kit per manufacturer recommendations. Eluted in nuclease-free water 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 Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer's protocols. Briefly, mRNA was purified from 200ng 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 using dUTP incorporation for strand marking. 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 cDNA 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). Single end reads of 100nt were generated for each sample using Illumina's HiSeq2500v4.