GSM8389556: LN95 cells, EPI-002, - androgen, biol rep 2; Homo sapiens; RNA-Seq
Cells were harvested in 1mL Trizol and total RNA was purified using the PureLink RNA mini kit (Invitrogen). 0.9 ug of RNA was used for library construction. Polyadenylated (PolyA+) RNA was purified using the NEBNext Poly(A) mRNA Magnetic  Isolation Module (E7490L, New England Biolabs) from 500 ng total RNA. First-strand cDNA was synthesized from heat-denatured purified mRNA using a Maxima H Minus First Strand cDNA Synthesis kit (Thermo-Fisher, USA) and random hexamer primers at a concentration of 200 ng/µL along with a final concentration of 40 ng/µL Actinomycin D, followed by PCR Clean DX (Aline Biosciences) bead purification on a Microlab NIMBUS robot (Hamilton Robotics, USA). The second strand cDNA was synthesized following the NEBNext Ultra Directional Second Strand cDNA Synthesis protocol (New England Biolabs) that incorporates dUTP in the dNTP mix, allowing the second strand to be digested using USERTM enzyme (NEB) in the post-adapter ligation reaction and thus achieving strand specificity.  cDNA was fragmented to by Covaris LE220 sonication to achieve 250-300 bp average fragment lengths. The paired-end sequencing library was prepared following the BC Cancer Genome Sciences Centre strand-specific, plate-based library construction protocol on a Microlab NIMBUS robot (Hamilton Robotics, USA).