GSM7928804: IPH3_FBY2567_1; Schizosaccharomyces pombe; ChIP-Seq
After quenching the solution with glycine (final concentration of 360 mM), cells were washed twice with a cold Tris-buffer saline (20 mM Tris-HCl [pH 7.5] and 150 mM NaCl) and frozen in liquid nitrogen. Cell pellets from 50 ml cultures were thawed and resuspended in 500 µL of cold lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100 and 0.1% Na-deoxycholate) that was supplemented with a protease inhibitor cocktail (1X PMSF and 1X PLAAC) and disrupted vigorously with glass beads in a FastPrep-24TM (MP biomedical) for 3 cycles of 30 sec at 6.5 m/s. Then, 150 µL of cold lysis buffer with proteases was added to increase the volume and samples were sonicated for 12 cycles of 10 sec at 20% amplitude with a Branson digital sonifier. Sonicated chromatin was then incubated overnight at 4 °C with 50 µl of magnetic beads IgG Dynabeads (Life Technologies, 11041) in the case of TAP tagged proteins, or Dynabeads M-280 Sheep Anti-Rabbit IgG (Invitrogen, 11203D) beads coupled with 2 ug of rabbit anti-Histone H3 total (ab1791, abcam), H3K36me3 (ab9050, abcam) or H3K9ac (06-942, Millipore Sigma). Beads were then washed twice for 4 minutes at 4oC with 1 mL of cold lysis buffer, twice with 1 mL of cold lysis buffer with 500 mM NaCl, twice with 1 mL of cold wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate and 1 mM EDTA), and once with 1 mL of cold Tris-EDTA (TE: 10 mM Tris-HCl [pH 8.0] and 1 mM EDTA [pH8.0]). Bound material was eluted by resuspending the beads in 50 µl elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA and 1% SDS) at 65 °C for 15 min at 1200 rpm in an Eppendorf Thermomixer. After a short centrifugation, reverse-crosslinking was performed by incubating eluted chromatin with 120 µl of TE and 1% SDS or 5 µl of WCE in 165 µl of TE with 1% SDS at 65 °C overnight. For ChIP-Seq experiments, reverse-crosslinking was perfomed by incubating eluted chromatin with 20 µl of WCE in 150 µl of TE and 1% SDS. Samples were treated with a proteinase K mix (150 mg proteinase K, 60 mg glycogen in TE) and DNA was extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1, [pH8.0], Invitrogen) and once with chloroform (Bioshop). Following ethanol precipitation, pellets were resuspended in 30 µl of TE plus 10 µg of RNAse A and incubated 1 h i at 37 °C. RNase-treated DNA was then purified with a QIAGEN PCR purification kit. The input DNA was diluted 400 times while the coimmunoprecipitated DNA was diluted 100 times in water. DNA were analyzed on a LightCycler 96 Instrument system (Roche) with the addition of the 2X SYBR Green in the presence of 0.15 μM of gene-specific oligonucleotide ChIP-sequencing libraries were prepared using the SPARQ DNA Sample Prep Kit Illumina Platform (Quantabio) according to the manufacturer's instructions.