Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: The pellets were thawed on ice, resuspended in 500 µl zymolyase solution (1.2 M sorbitol, 10 mM Tris-HCl pH 8, 10 mM CaCl2, 1% v/v beta-mercaptoethanol, 0.1 U/µl zymolyase (Zymo Research)) per pellet and incubated at 37 °C for 30 minutes with shaking at 500 rpm to digest the cell walls. The resulting spheroplasts were isolated by centrifugation for 5 min at 3500xg. Spheroplasts were resuspended in 540 µl lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1% w/v sodium deoxycholate, 1 mM PMSF, 1 mM benzamidine, 1:100 yeast protease inhibitor cocktail (Sigma Aldrich, P8215)). The lysates were sonicated using the Covaris ME220 instrument in 130 µl screw-cap microtubes for 3 minutes (PIP = 75, DF = 15%, 1000 cycles per burst, 9 °C). Sonicated lysates were centrifuged at 17000xg for 10 minutes to pellet insoluble material. 5 µl aliquots from each sample were decrosslinked to check the size of chromatin fragments, which was between 100-500 bp. The amount of chromatin in each sonicated sample was estimated from the DNA concentration in the decrosslinked aliquot. For the IP, equal amount of chromatin from each sample was taken based on this estimate, and S. pombe sonicated chromatin was spiked in to at approximately 1% relative to that amount (S. pombe chromatin was obtained in the same way as above, except with two times larger volume of zymolyase solution and twice as long incubation time). Volumes were adjusted with lysis buffer to be equal between samples. 250 µl of Dynabeads Pan Mouse IgG (Thermo Fisher) per sample were washed 3 times with PBS/BSA (PBS with 5 mg/ml bovine serum albumin (Sigma Aldrich)), resuspended in the sonicated chromatin solution, and rotated overnight at +4 °C. They were washed three times each with lysis buffer, lysis buffer containing 500 mM NaCl, and LiCl wash buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and once with elution buffer (50 mM Tris-HCl pH 8, 1% SDS, 10 mM EDTA). Chromatin was eluted from beads in elution buffer at 65 °C with shaking for 20 minutes. Eluted material was diluted five-fold to reduce the concentration of SDS to 0.2%, then 1:100 RNase cocktail (Thermo Fisher, AM2286) was added and the samples were incubated at 37 °C for 45 minutes to digest RNA. SDS was added to a total of 0.5% and Proteinase K to 0.5 mg/ml (Thermo Fisher, AM2546), and the samples were shaken overnight at 65 °C to reverse crosslinked DNA. In parallel, input control samples (10 µl sonicated chromatin) were subjected to RNA digestion and reversion of crosslinking in the same way. Next day, the DNA was purified with the QIAquick Gel Extraction kit (QIAGEN) and eluted into 50 µl water. Sequencing libraries were prepared using the NEBNext Ultra™ II DNA Library Prep Kit (NEB). 4 PCR cycles were used for the input samples and 17 cycles for the IP samples. The resulting libraries were pooled and dual size-selected with 0.6X and 1.2X AMPure XP (Beckman Coulter) bead ratios. The pool was sequenced on NextSeq 550, High Output kit (Illumina), paired-end with a read length of 38 bases.