3.3.1 Molecular cloning
H2B and K120R constructs used in this study were created by amplification of synthetic H2B sequence using primers listed in 2.3.4.4 and Phusion DNA polymerase. Obtained products and pcDNA5/TO vector were digested by restriction enzymes followed by ligation with T4 DNA ligase and clone selection on Ampicillin agar plates. For expansion constructs were transformed into E. coli XL1-Blue strain followed by culturing in Ampicillin-containing 2YT medium.
3.3.2 RNA isolation
RNA isolation was performed with QIAzol® reagent according to manufacturer’s instructions. Cells were washed with PBS, lysed by addition of 1ml of QIAzol® reagent to each well (6-well format) and collected into 1.5 ml tubes. After addition of 200 µl of chloroform samples were vortexed and centrifuged at 10,000g for 20 min (4°C). After that the aqueous phase was collected and chloroform extraction was performed a second time followed by overnight isopropanol precipitation at -20°C. After that samples were centrifuged at maximal speed for 20 min (4°C), pellets were washed twice with 70% ethanol, dried on vacuum concentrator and re-dissolved in 50 µl of DEPC water. RNA concentration was measured using a NanoDrop.
3.3.3 cDNA synthesis
For DNA synthesis 1 µg of total RNA was mixed with 2 µl of 15 µM random primers and 4 µl of 2.5 mM dNTP mix and incubated 5 min at 70°C. After that 4 µl of reverse transcription master mix containing 2 µl 10× reaction Buffer, 10 units of RNAse Inhibitor, 25 units of Reverse Transcriptase and 1.625 µl of DEPC water were added to each sample. cDNA synthesis was performed at 42°C for 1 h followed by enzyme inactivation for 5 min at 95°C.
Finally, samples were brought to 50 µl volume by DEPC water.
41 3.3.4 Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed in final volume of 25 µl. For each reaction 1 µl of cDNA or ChIP DNA was mixed with 8.5 µl of ddH2O, 1.5 µl of 5 µM primer mix and 14 µl of qRT-PCR mix containing 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween-20, 3 mM MgCl2, 200 μM dNTPs, 20 U/ml Taq Polymerase, 0.25% Triton X-100, 1:80,000 SYBR Green I and 300 mM Trehalose.
The PCR reaction was performed using two-step protocol:
95 °C 2 min
95 °C 15 s }
40 cycles 60 °C 1 min
The PCR reaction was followed by a melting curve recording from 70 °C to 95 °C with the plate read every 0.5 °C. All qRT-PCR samples were normalized to an internal reference gene (HNRNPK) and displayed relative to the control non-differentiated sample. Statistical analysis was done with ANOVA test.
3.3.5 Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed as described (Karpiuk et al., 2012).
Briefly, cells were crosslinked by 1% formaldehyde in PBS for 1 min. The reaction was quenched by addition of 1 ml 1.25M glycine for 5 min. After that cell were washed with ice- cold PBS and scraped in Nelson Lysis Buffer with inhibitor cocktail. Obtained nuclei were centrifuged at 12,000g for 1 min (4°C), washed with Nelson lysis buffer and resuspended in 300 µl of Gomes Lysis buffer in the presence of 1% w/v SDS. Sonication was performed with Bioruptor at high power settings three times 10 min with 10s pulses followed by 10s pause.
After that samples were pre-cleared with 100 µl of Sepharose 4B (50% slurry in Gomes Lysis Buffer) for 1h at 4°C, diluted, aliquoted, snap frozen in liquid nitrogen and stored at -80 C until further use.
One aliquot was taken for each antibody. Chromatin was diluted to 1 ml with Gomes Lysis Buffer without SDS and with inhibitor cocktail and an appropriate amount of antibody was added (see Materials section). Samples were incubated with the antibodies overnight at 4°C, then 30 μl of Protein-G Sepharose (50% slurry in Gomes Lysis Buffer with inhibitor cocktail) were added and incubated for another 2 h at 4°C. After centrifugation (2000g, 2 min, 4°C) beads were washed three times with Gomes Lysis Buffer with inhibitor cocktail and without
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SDS, three times with Gomes Wash Buffer with inhibitor cocktail and twice with TE-buffer.
Reverse crosslinking was performed by adding 100 μl of 10% (w/v) Chelex 100 slurry and incubating at 95 °C for 10 min. After that 2 μl of Proteinase K (20 μg/μl) were added to each sample, followed by the incubation at 55°C for 30 min. Proteinase K was inactivated by heating the samples to 95 °C for 10 min. After that samples were centrifuged at 12,000g for 1 min at 4°C and the supernatant containing DNA was used for quantitative real-time PCR.
The background binding was determined by performing a ChIP with a non-specific IgG antibody. ChIP inputs preparation: 5 µl (10% relative to ChIPs) of chromatin extracts were precipitated by adding 100% EtOH and 1 μl of Pink Precipitant (5 mg/ml) and incubating overnight at -20°C. The pellets were washed twice with 70% EtOH, dried and processed with Chelex addition as described above for ChIP samples. ChIP samples were normalized to input DNA samples, and displayed as “% of input”. Statistical analysis was done with ANOVA test.
3.3.6 Microarray
Total RNA for microarray experiments was isolated as described in 3.3.1 and Illumina whole-genome gene expression analysis using a human HT-12 v4 beadchip was performed by the Vancouver Prostate Centre Laboratory for Advanced Genome Analysis, Vancouver, Canada.
Gene expression data was analyzed by Frank Krammer, WG Statistical Bioinformatics, University of Goettingen, using log2 transformation and quantile normalization of expression levels (Bolstad et al., 2003). Background correction was applied according to the manufacturer’s advice. In order to determine significant differences of expression levels between the different groups a moderated Student’s t-test was computed on a gene-by-gene basis using the empirical Bayesian statistics in the ‘limma’ package (Smyth, 2004). P-values were adjusted for multiple testing using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995) to avoid a high number of false positives and to stay below a false discovery rate of 5%. All analyses were performed using the free statistical software R (version 2.12.2) (RDev, 2011).
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