title: GSM3324101: U2OS+As_UNR_Rep1; Homo sapiens; RNA-Seq
library_construction_protocol: 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.
attributes.tag: GEO Accession
attributes.value: GSM3324101