E-MTAB-2368:SO_974_974_WCE
Illumina Genome Analyzer IIx sequencing; heat shock SUMO-2 ChIP-seq
heat shock SUMO-2 ChIP-seq
untreated_input
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. 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 2.1 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 14 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, a fraction corresponding to 90% of the lysate volume was incubated with 21 ug SUMO-2 antibody and subsequently with protein G sepharose 4B beads (Sigma) for 1 h using a final bead bed volume of 150 ul. To reduce non-specific binding, beads had been pre-incubated with 0.5% (w/v) BSA in PBS overnight. 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_974_974_WCE
Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, United Kingdom
E-MTAB-2368:SO_974_974_WCE
Experimental Factor: growth condition
untreated
Experimental Factor: immunoprecipitate
input
Experimental Factor: beads
none