sin, in cubated for 1 h rotating, and washed three times with lysis buffer. Washes were removed through centrifuga tion of the HaloLink resin at 1000 ��g for 5 min and as piration. At the final wash, the resin was resuspended in cleavage buffer and rotated for 2 h at room temperature. Resin was centrifuged at 2000 x g for 5 min and super natant removed. TEV protease was removed by the addition of HisLink resin to the supernatant and incuba tion for 20 min rotating at room temperature. HisLink was removed through centrifugation at 1000 �� g for 5 min and the resulting supernatant snap frozen in liquid nitro gen and stored at ?80 C. Quantification of the protein was carried out using BCA Protein Assay. Purification was confirmed through Western blot analysis using rabbit anti BORIS antibody.
Western blot analysis Protein extracts or precipitated protein complexes were separated on a 4 12% gradient NuPAGE polyacrylamide gel and then blotted onto nitrocelluose membrane as described by Jones et al. After incubation with blocking solution the membrane was incubated with corresponding anti bodies overnight at 4 C. After several washes, bands were revealed with the corresponding horseradish perox idase coupled secondary antibody and detected using the ECL detection kit according to the manufacturers protocol. Densitometry scanning of the intensity of bands on the Western blot was quantified using ImageJ. The p values were obtained using one way ANNOVA test after intensity values were normalised to GAPDH levels.
In vitro binding assay For RNA and DNA binding assays, 1 mg of purified BORIS protein was incubated with 125 nM of each bio tinylated homopolymer AV-951 in 400 ml of Binding Buffer, 1 mM dithio threitol and 0. 2% NP 40 at 4 C overnight. Nucleo tide,protein complexes were isolated by addition of 20 ml prewashed Dynabeads M280 Streptavidin to the reaction for 30 min rotating at room temperature. Complexes were magnetically captured and washed three times in RBB. After the final wash, beads were resus pended in 10 ml NuPAGE LDS sample buffer supple mented with 5 mM DTT, heated to 70 C for 5 min. Captured proteins were resolved by 4 12% SDS PAGE and analysed by Western blot using anti BORIS antibody. Analysis of microarray data Affymetrix Expression array files were analysed using Partek software, version 6. 5 Copyright ? 1993 2010.
Principle component analysis was applied to identify any independent sources of variation in the data. We compared data for BORIS bound RNA transcripts with genome wide gene expression profiles for each selected cell type with at least two biological replicates. A t test was performed and transcripts were considered to be prefer entially associated with BORIS when the signals from the immunoprecipitated RNA fractions were enriched more than 2 fold, with a p value 0. 01. The gene expres sion data have been deposited in NCBIs Gene Expres sion Omnibus and are accessible through GEO series accession number GSE42294. Pathway analysis an