GSM4849162: RNAseq_pA-_Ery_iPSC_WT_SBAD02_rep2; Homo sapiens; RNA-Seq
isolation protocol: 1-5x10e6 cells were fixed in 1 ml TRI-reagent (Sigma), snap frozen and stored at -80ºC. RNA was isolated from 5×106 day 21 erythroid cells using the Direct-zol RNA kit (Zymo) as per manufacturer's instructions. DNase I digestion was performed on column. RNA purity was assessed using Nanodrop ND-1000 spectrophotometer (ThermoFisher Scientific). RNA integrity was analysed using RNA ScreenTape Assay (Agilent) 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.