Approximately 1-3 X 10^7 cells were fixed by addition of 1% formaldehyde to cell media for 10 min at RT with gentle agitation. Formaldehyde was quenched by addition of glycine to 0.125 M with gentle agitation for 5 min at RT. Cells were pelleted at 2000 xg, washed twice in PBS, and resuspended in 0.8 mL ice–cold cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) supplemented with Complete protease inhibitors (Roche) and RNasin (Promega), incubated on ice 10 min, and pelleted by centrifugation at 2000 xg for 5 min at 4°C. Next, the supernatant was removed and pellets were resuspended in 1 mL nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA.Na2, 1% SDS, RNasin (Promega)) and incubated for 10 min at 4°C. Afterwards, 0.5 mL of IP dilution buffer was added (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS), and chromatin was fragmented to an average size of 300 bp by using PicoBioruptor (Diagenode) using 10 cycles of 30 s on plus 30 s off, maximum output. Samples were centrifuged at max speed for 10 min at 4°C, and the supernatant (sheared chromatin) was saved at -80°C. Chromatin was diluted to 1:5 with IP buffer, and the assayed antibody in addition to 50 μL of prewashed protein A/G 50% slurry, preblocked for one hour with 0.5% of tRNA and 0.2% BSA, was added and rotated overnight at 4°C. The next day, beads were washed first three times with low salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 150 mM NaCl, 1% Triton X-100, 0.1% SDS); next three times with high salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS); finally two times with LiCl buffer (10 mM Tris-HCl pH 8, 1 mM EDTA.Na2, 250 mM LiCl, 1% NP-40, 1% DOC). After the last LiCl wash, beads were resuspended in 100 μL of 50mM Tris-Cl pH 7.0, 5 mM EDTA and 1% SDS and de-crosslinked at 65°C for 2 h. Next, beads were treated with DNAse I for 15 min at 37°C, and total RNA was extracted with TRIzol (Invitrogen). RNA-seq libraries were prepared with Ovation RNA-seq Systems 1-16 for Model Organisms (Nugen).
GEO Accession
GSM3263450