NextSeq 500 paired end sequencing; ChIP-seq of histone H3 and H3K9me2 in WT and dbl2 mutant fission yeast
ChIP-seq of histone H3 and H3K9me2 in WT and dbl2 mutant fission yeast
At OD 0.5 yeast cultures were fixed by adding formaldehyde to a final concentration of 1 %. After 30 min of shaking at room temperature, the remaining formaldehyde was quenched by 125 mM glycine for 10 min at room temperature with shaking. Cells were washed with PBS and broken with glass beads in the Lysis Buffer (50 mM Hepes pH 7.6, 1 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, FY protease inhibitors [Serva], 1 mM PMSF) using the FastPrep24 machine (MP Biomedicals; 9x 20 sec at 5.5 m/s). Extracted chromatin was sheared with the Bioruptor sonicator (Diagenode) using 30 cycles of 30 sec ON, 30 sec OFF on high power settings.  Four hundred millilitre cultures of S. pombe WT and dbl2 knock-out cells were grown to exponential phase in complex YES medium at 32°C. For each immunoprecipitation (IP) in a given biological replicate, the same amount of chromatin extract was used (2.27 mg and 2.48 mg total protein, respectively), and 1/10 of chromatin extract amount was kept for input DNA control. For each IP, 5 μg of antibody (H3: Ab1791, H3K9me2: Ab1220, both Abcam) were incubated with chromatin extract for 1 hour at 4°C. Then, samples were transferred to 50 ul of BSA-blocked Protein A-coated magnetic beads (Thermo Fisher Scientific, 10002D) and incubated for further 4 hours at 4°C. The precipitated material was washed twice with Lysis Buffer (50 mM Hepes pH 7.6, 1 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), Lysis 500 Buffer (50 mM Hepes pH 7.6, 1 mM EDTA pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl/NP-40 Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate), once in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), and eluted in Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). The washed precipitated material and input chromatin extract were decrosslinked for 6 hours at 65°C, treated with RNase A (Thermo Fisher Scientific) followed by proteinase K (Roche). DNA was extracted using phenol-chloroform extraction and sodium acetate/ethanol precipitation. DNA was further purified on SPRIselect beads (Beckman Coulter, B23317) to remove RNA and small DNA fragments. The concentration of input DNA was measured by Quantus and fragment size distribution was checked on Agilent Bioanalyzer using the High Sensitivity DNA Assay. The Illumina TruSeq Nano DNA kit with Illumina TruSeq DNA CD Indexes were used for library construction using the manufacturer's instructions, with the following exceptions: no initial DNA fragmentation was performed; after end-repair DNA was purified using 4 volumes of AMPure Beads.
Experimental Factor: genotype
dbl2 knockout
Experimental Factor: immunoprecipitate
anti-H3K9me2