The cleared supernatant was incubated with 10 ug BORIS antibody coupled to dynabead protein A for 1 Veliparib chemical structure 2 hours at 4 C. After extensive washes with buffer D, 0. 1 U ml of RNaseOut, 0. 02% NP 40 and 0. 25% Triton X 100 the bead protein complex was incubated with 50 units of DNase 1 containing 100 units of RNase OUT for 5 minutes at 37 C. An equal volume of pro teinase K containing buffer was added and incubated for another 15 minutes at 37 C. RNA was extracted with standard phenol chloroform procedure and precip itated with 2 ul of glycogen. The RNA was used for either hybridization to Affyme trix U133 plus 2. 0 expression arrays or for RT qPCR verification of BORIS target transcripts. For array ana lysis, double stranded cDNA was synthesized from 1.
5 5 ug total RNA using the Affymetrix One cycle cDNA synthesis kit following the manufacturers instructions. Synthesis of Biotin labeled cRNA was per formed using the Affymetrix GeneChip IVT labeling kit followed by purification with the sample cleanup mod ule. Labeled cRNA was then fragmented and hybridized to Affymetrix GeneChip Human Genome U133Plus 2. 0 arrays overnight. Hybridisation and scanning was performed in house at Barts Cancer Institute. For RT qPCR analysis, RNA in the IP material was reverse transcribed to cDNA using superscript III following the manufacturers instructions. Quantitative real time PCR was performed on ABI7500 equipment using gene specific primer pairs and amplification condi tion of 2 min at 50 C, 10 min at 95 C, and then 40 cycles of 15 secs at 95 C and 45 secs at 60 C.
Total RNA was isolated using silica based spin column extraction kit follow ing the manufacturers protocol. Total RNA was treated with RNase free DNase1 to reduce genomic DNA contamination. RNA integrity was evaluated using the Agilent Bioanalyzer. Two micrograms of total RNA was reverse transcribed with SuperScriptase III using Oligo dT primers or random hexamers ac cording to the manufacturers protocol. Negative controls contained RNase free water substituted for re verse transcriptase. Recombinant BORIS purification The mammalian expression plasmid pM49 T4738 car ries BORIS with an N terminal HaloTag. Adherent HEK293T cells were transfected using Lipofectamine 2000 using standard methods. Cells were cultured for 48 h prior to harvest. Media were aspirated and cells washed in cold PBS before removal by cell scraping.
Cells were centrifuged at 2000 �� g for 5 min. The cell pellet containing over expressed HaloTag BORIS was stored at ?80 C overnight. The cell pellet was lysed in lysis Anacetrapib buffer supplemented with BaculoGold protease inhibitor. HaloTag BORIS was purified as per manufacturers protocol. The cell pellet was lysed on ice in 1 ml of lysis buffer per 2 �� 107 cells for 10 minutes, followed by 5 min pulse sonication using Diagenodes Bioruptor 3 min. Crude lysate was centrifuged at 10,000 �� g for 30 min.