GSM1895989: Input DNA; Homo sapiens; ChIP-Seq
For each ChIP experiment, material corresponding to 5x106 starting cells was used. Samples were diluted with an equal volume of D-Buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1% Triton X-100). 5 µg of antibody (FUS sc-47711 Santa Cruz and EWS sc-48404 Santa Cruz) or 2 µg antibody for acetylated histone protein (ac-H3K9 ab4441-50 Abcam) were used for each ChIP. Samples were incubated in an ultrasonic water bath for 25 min, at 4°C, and afterwards centrifuged at 12,000 g for 10 min, at 4°C. 20 µl magnetic AG-beads (Invitrogen) were used for each ChIP. Beads were washed 3 times in 1 ml of supplemented IP-Buffer before use. After the final wash, beads were suspended in 40 µl of supplemented IP-Buffer and mixed with 90% of the chromatin supernatant described above. The mix was incubated in rotation overnight, at 4°C. Beads were washed twice with 200 µl of ice-cold supplemented IP-Buffer and three times with 200 µl ice-cold D-Buffer supplemented with protease inhibitors. Cross-linking was reversed by adding Reverse X-link Buffer and Protease K (Invitrogen) and incubating in water bath for 20 min, at 55°C. The remaining 10% of the reversed cross-linking chromatin supernatant was used as input. Supernatants were transferred to fresh tubes and incubated at 95°C to inactivate Protease K. DNA was purified using DNA Purification Buffer plus DNA purification magnetic beads, as recommended (Invitrogen). DNA was stored at -20°C. DNA from FUS, EWS, and Ac-H3K9 ChIP experiments, as well as the corresponding input, was sequenced using the Illumina Hiseq 2000 platform at BGI in Shenzhen, China. Library preparation, cluster generation and sequencing by synthesis were performed according to manufacturer's protocol. All raw reads were aligned using Burrows-Wheeler Aligner (BWA version 0.5.8c (r1536) [48]) to the human reference genome (hg19). Aligned reads were processed by Model-based Analysis of ChIP-Seq (MACS) 1.4.0rc2 [49] for peak calling. Significant peaks were defined using the criteria of a threshold of minimally 9 reads and p-value less than 10-8 as suggested previously [50]. Input ChIP DNA was used as negative control.