GSM3580871: RNAseq_rs10758656_G_d10_clone1E4; Homo sapiens; RNA-Seq
RNA was extracted by addition of 0.1 ml 1-bromo-3-chloropropane, pipette mixing and separation in a Phase Lock gel Heavy tube (5Prime) and then precipitation with 1 µl of GlycoBlue and an equal volume (~500 µl) isopropanol and centrifugation (10 min, 12,000 rcf, 4 ºC). The RNA pellet was washed with 75 % ethanol, resuspended in DEPC-treated water, and stored at -80 ºC for less than one year. For RNA-seq total RNA was treated with Turbo DNAse (Invitrogen) at 25 ºC for 60 min, then RNA was separated using phenol-chloroform isoamylalcohol and a PhaseLock Light-gel tube (5Prime).  Treated RNA was precipitated at -80 ºC overnight with sodium acetate, glycoblue, and 75 % ethanol, before centrifugation (12,000 rcf, 4 ºC), 75 % ethanol wash and resuspension in DEPC-treated water. Globin and rRNA sequences were depleted from up to 5 µg of treated RNA using Globin-Zero Gold (Illumina), before PolyA selection with NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs), and indexing with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturers' instructions.