title: GSM3044985: Human_436_20B_UNC_d4_2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted using the qiaquick purification kit RNA samples were quantified by qubit (Life Technologies) and quality by Agilent Bioananlyzer. Two hundred nanograms Total RNA from 42 samples were library prepared using TruSeq Stranded Total RNA kit (Illumina). RNA samples were ribosomal RNA depleted using Ribo-zero Gold rRNA beads, following purification the RNA was fragmented. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using RNase H and DNA Polymerase I. A single “A” based were added and adapter ligated followed by purification and enrichment with PCR to create cDNA libraries. Final cDNA libraries were size validated using Agilent Bioanalyzer and concentration validated by qPCR (Kapa Biosystems/Roche). All libraries were normalized to 10nM and pooled together, denatured with 0.2N NaOH and diluted to a final concentration of 1.4pM.  1.3ml of 1.4pM pooled libraries were loaded onto an Illumina NextSeq cartridge for cluster generation and sequencing on an Illumina Nextseq500 instrument (Illumina) using Paired-end 75bp protocol to achieve ~ 40 million reads per sample