GSM1632423: BJ-OSK-B; Homo sapiens; RNA-Seq
Cells were harvested with Trizol reagent and stored at -80 °C until use. Total RNA was extracted using the Direct-zolTM Miniprep kit (R2052). The standard Agilent SureSelect Strand-Specific RNA sequencing protocol was followed. Briefly, the polyA fraction was purified using two rounds of oligo dT-magnetic bead purification. The resulting polyA fraction was fragmented and first strand cDNA was done with random primers in the presence of Actinomycin D using standard techniques. The resulting first stand cDNA was purified using AMPure Beads and second strand cDNA and end repair was done using standard techniques.  The blunt ended double stranded cDNA was incubated with exo- E.coli DNA polymerase to add an adenosine to the ends for T/A cloning of the adaptors. PCR amplification was done in the presence of uracil DNA glycosylase to generate strand specificity.  A second round of PCR amplification was done to add unique 6-bp barcodes to each sample and add sequences necessary for flow cell attachment.  The resulting mRNA libraries were quantitated using qPCR following the manufacturer's instructions (Kapa Biosystems, Woburn MA).
GEO Accession
GSM1632423