GSM3208396: D5_DMSO; Homo sapiens; RNA-Seq
Total RNA extraction was performed according to the Qiagen miRNeasy kit. RNA concentrations were determined using Qubit fluorometric quantification and purity by the RNA 6000 Bioanalyzer kit (Agilent) RNA-Seq libraries were constructed with the Truseq stranded mRNA sample preparation (Low throughput protocol) kit from Illumina.    Poly-A based mRNA enrichment: 1.2µg of total RNA was used for the construction of the libraries.  Using poly-T oligo attached magnetic beads, 1,2µg of total RNA was purified in order to obtain only poly-A mRNA.    Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature.  The cleaved RNA fragments are copied into first strand cDNA using SuperScript IV reverse transcriptase, Actinomycin D and random hexamer primers. The Second strand cDNA was synthesized by replacing dTTP with dUTP. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with 15 cycles of PCR. The final cDNA libraries were validated with a Fragment Analyzer (Advanced Analytical, Ankeny, IA) and quantified with a KAPA qPCR kit (Kapa Biosystems, Wilmington, MA).    Library sequencing: On 8 sequencing lane of a flowcell V4, the 32 libraries were pooled in equal proportions by sets of 4 libraries per lane, denatured with NaOH and diluted to 16 pM before clustering. Cluster formation, primer hybridization and single-end read, 50 cycles sequencing were performed on cBot and HiSeq2500 (Illumina, San Diego, CA) respectively.