GSM3324100: U2OS+As_IgG_Rep3; Homo sapiens; RNA-Seq
Cells were washed four times in PBS and then suspended in polysome lysis buffer (0.5% v/v NP40, 100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH7.0) then incubated on ice for 5 min before being transferred to -80 °C for a period of no less than 8 h Finally, the raw lysates were thawed on ice, centrifuged at 15000 x g at 4 °C for 15 min, then the supernatants collected and the pelleted debris discarded. The lysate was further purified by a second centrifugation at 15000 x g at 4 °C to separate the aqueous lysate from unwanted lipids. To precipitate UNR and associated molecules, Protein A-conjugated Dynabeads (Life Technologies) were firstly incubated with either an antibody against UNR (polyclonal rabbit antibody, Novus Biologicals NBP1-71914) or a control IgG (Invitrogen 10500C) in PBS containing 0.01% (v/v) Tween-20 (PBST) in tubes attached to a rotating mixer for 1 h at room temperature. Following this, the beads were similarly blocked at 4 °C in 0.1% (w/v) bovine serum albumin (BSA) in PBS. The beads were then washed twice in ice-cold PBST and then once in ice-cold NT2 buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1mM MgCl2; 0.05% [v/v] NP-40) before resuspension in NT2 containing 0.5 % (v/v) RNase OUT, 400 μM VRC and 1 mM DTT. Cleared lysate  was then added at 1 part to 9 parts bead suspension and mixed by rotation for 4 h at 4 °C. The beads were then washed five times in ice cold NT2 and each sample divided, 4 parts for RNA analysis RNA was extracted from immune complex-loaded beads using Trizol (Life Technologies) according to the manufacturer's instructions. RNA-Seq cDNA libraries were then made using the low sample protocol from the Illumina TruSeq RNA sample preparation v2 guide, except that the oligo-dT selection step was omitted, where possible 100 ng of RNA was used per sample, and libraries were quantified using Qubit Fluorometry.  The libraries were normalised to 10 nM.
GEO Accession
GSM3324100