E-MTAB-2368:SO_1308_1308_37-2
Illumina Genome Analyzer IIx sequencing; heat shock SUMO-2 ChIP-seq
heat shock SUMO-2 ChIP-seq
untreated_SUMO2_ChIP_protein G Dynabeads beads
Human osteosarcoma (U2OS) cells were obtained from CRUK and grown at 37C according to standard cell culture protocols. U2OS cells were grown at 37C and either left untreated or incubated at 43C for 30 minutes to induce heat shock. Bead-bound protein-DNA complexes were washed twice in approximately 14 ml of each wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 150 mM NaCl), wash buffer II (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 500 mM NaCl) wash buffer III (0.25 mM LiCl, 1 % NP-40, 1 % sodium deoxycholate, 1 mM EDTA pH 8.0, 10 mM Tris pH 8.1) and TE buffer (10 mM Tris pH 8.1, 1 mM EDTA pH 8.0) and eluted into 2.5 ml elution buffer containing 1% SDS and 0.1 M sodium bicarbonate for 20 min at 30C while shaking. An equal volume of TE buffer was added to eluates prior to digestion of RNA by incubation with 0.2 mg/ml RNase A (Fermentas) at 37C for 2 hours.  Reversal of cross-links was carried out by incubation in the presence of 0.2 M sodium chloride overnight at 65C, followed by incubation with 0.25 mg/mL proteinase K (Roche), 40 mM Tris pH 6.5 and 10 mM EDTA for 1 h at 55C. DNA was purified using a PCR purification kit (Qiagen).  To derive genomic DNA to be used as an input reference for SUMO-2 ChIP-seq, a fraction corresponding to the sonicated, diluted and cleared whole cell extracts that had been prepared for SUMO-2 ChIP was removed. These input samples were treated for removal of RNA and cross-link reversal and DNA was extracted as described above. Approximately 3.5-4.5 x 10^7 U2OS cells were used per treatment. Cells were chemically cross-linked by adding formaldehyde solution directly into cell culture medium to a final concentration of 1 %. Cells were fixed by incubation at room temperature for 10 min, followed by incubation with 0.125 M glycine for 5 min. Cells were washed twice and scraped into ice-cold PBS. Cell pellets were flash frozen in liquid nitrogen and stored at -80C. Frozen cell pellets were lysed in 1.8 ml lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris (pH 8.1), and protease inhibitors. To shear chromatin to fragments of about 200-500 bp size, samples were sonicated in 300 _l volumes for 15 cycles (7.5 min total sonication time) at high setting using a Bioruptor (Diagenode). Sonicated lysates were then cleared by centrifugation for 10 min at high speed, diluted 1/10 with dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris at pH 8.1) and incubated with SUMO-2 antibody (Life Technologies, 51-9100) and 0.1% Brij-35 overnight at 4C. In detail, to capture protein-DNA complexes, lysates were incubated with 13 ug SUMO-2 antibody and subsequently with protein G Dynabeads (Life Technologies) for 1 h using an equivalent of 300 ul bead solution. To reduce non-specific binding, beads had been pre-incubated with 0.5% (w/v) BSA in PBS overnight.  Bead-bound protein-DNA complexes were washed twice in approximately 13 ml of each wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 150 mM NaCl), wash buffer II (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 500 mM NaCl) wash buffer III (0.25 mM LiCl, 1 % NP-40, 1 % sodium deoxycholate, 1 mM EDTA pH 8.0, 10 mM Tris pH 8.1) and TE buffer (10 mM Tris pH 8.1, 1 mM EDTA pH 8.0) and eluted into 2.3 ml elution buffer containing 1% SDS and 0.1 M sodium bicarbonate for 20 min at 30C while shaking. An equal volume of TE buffer was added to eluates prior to digestion of RNA by incubation with 0.2 mg/ml RNase A (Fermentas) at 37C for 2 hours.  Reversal of cross-links was carried out by incubation in the presence of 0.2 M sodium chloride overnight at 65C, followed by incubation with 0.25 mg/mL proteinase K (Roche), 40 mM Tris pH 6.5 and 10 mM EDTA for 1 h at 55C. DNA was purified using a PCR purification kit (Qiagen). Libraries were constructed using approximately 10 ng purified DNA.  Single-end libraries were generated using either the Illumina ChIP-seq DNA sample Preparation Kit (IP-102-1001) or the TruSeq DNA sample preparation kit (PE-940-2002). In brief, ChIP or input DNA was end repaired and A-tailed before adaptors were ligated on the fragments. The ligated samples were then size selected on a 2 % low melting agarose gel to approximately 250-300 bp. An 18 cycle PCR was performed and libraries were quantified using a Nanodrop Spectrophotometer. Library quality was validated by High Sensitivity Bioanalyzer ChIP (Agilent).
CENTRE FOR GENE REGULATION AND EXPRESSION, UNIVERSITY OF DUNDEE, DUNDEE DD1 5EH, UNITED KINGDOM
E-MTAB-2368:SO_1308_1308_37-2
Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, United Kingdom
E-MTAB-2368:SO_1308_1308_37-2
Experimental Factor: growth condition
untreated
Experimental Factor: immunoprecipitate
SUMO2_ChIP
Experimental Factor: beads
protein G Dynabeads