title: GSM7678405: tof1_M_H2AP_rep2; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: 25 ml of cultures were used per each antibody used for ChIP. Cells were washed and resuspended in YP media, then 1% formaldehyde (Sigma-Aldrich) was added, and cells were incubated for 45 min at 25°C. To quench the formaldehyde 125 mM glycine (Alfa Aesar) was added followed by 5 min incubation. Cells were then washed with cold PBS, pelleted and frozen in liquid nitrogen. For a subset of experiments (Spiked with Schizosaccharomyces pombe) normalization by adding Schizosaccharomyces pombe (S. pombe) cells containing the HO-endonuclease inducible system (AW507). Primary cultures of AW507 (described in Watson et al., Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus, Gene, 2011) were grown in EMM media supplemented with leucine (100 μg/ml) at 30°C overnight, then kept at logarithmic phase for a day, before diluting to a secondary culture to reach logarithmic phase (5x106 cells) again the next day. Cells were then pelleted and re-suspended in pre-warmed EMM supplemented with leucine, histidine and uracil (100 μg/ml each) to induce HO endonuclease (Purg1loxON). After two hours of incubation at 30°C cells were fixed and collected as described above for S. cerevisiae cells. Pellets from liquid nitrogen were resuspended in 500 μl SDS buffer (1% SDS, 10 mM EDTA, 5M Tris HCl, cOmplete Tablets, Mini EDTA-free EASYpack (Roche), PhosSTOP (Roche)), then lysed in a FASTPREP machine, 5 rounds of 1 min at 6.5 power, with 200 μl of 0.5 mm silica beads. Silica beards were then separated and discarded and IP buffer (0.1% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM TRIS HCl (pH8), cOmplete Tablets, Mini EDTA-free EASYpack (Roche), PhosSTOP (Roche)) was added to the lysate to a final volume of 1 ml. Focused-Ultrasonicator (Covaris, M220) was then used to sonicate the samples (Average incident power – 7.5 Watts, Peak Incident Power – 75 Watts, Duty Factor – 10 %, Cycles/Burst – 200, Duration – 20 min). For 50 ml starting culture the supernatant was diluted to 5 ml in total. 50-50 μl of protein A Dynabeads (Invitrogen) and protein G Dynabeads (Invitrogen) were mixed and washed 3 times in IP buffer then added to the samples and was incubated for 2 h at 4°C. After discarding the beads 2-2 ml of the supernatant was incubated with either H2A 1:500 (active motif) or 1.6 μg/ml H2AP (Abcam) on a rotating wheel at 4°C for 15 – 20 h. The rest of the sample was kept for input. If RFA1 ChIP-SEQ was also performed, the volume of the starting culture was increased to 75 ml and the sample after sonication was diluted to 7.5 ml in total and 75-75 μl of protein A and G Dynabeads were used to reduce unspecific binding and RFA1 antibody (1:10000, Agrisera) was added to 2 ml of the pre-cleaned sample followed by incubation on a rotating wheel at 4°C for 15 – 20 h. 30-30 μl of protein A and G Dynabeads were washed 3x with IP buffer and was added to each antibody reaction and incubated at 4°C for 4 h. Beads were then washed at 4°C for 6 min in TSE-150 (1% Triton-X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris HCl (pH8), 150 mM NaCl), followed by TSE-500 (1% Triton-X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris HCl (pH8), 500 mM NaCl), followed by LiCl wash (0.25 M LiCl, 1% NP-40, 1% dioxycholate, 1 mM EDTA, 10 mM Tris HCl (pH8)) and finally Tris-EDTA (TE pH8). Samples were then eluted in 400 μl elution buffer, for 30 min at room temperature. Input samples were prepared from 50 μl starting material mixed with 150 μl of elution buffer (200 μl final volume). For reverse crosslinking and protease treatment NaCl (500 mM final concentration) and proteinase K (Invitrogen, 500 μg/ml final concentration) was added and incubated at 65°C overnight. RNase treatment was then performed by adding DNase-free RNase (Roche, 25 μg/ml final concentration) followed by incubation at 37°C for 30 min. DNA was then purified with Qiagen PCR purification kit. NGS libraries were prepared using NEBnext Ultra II library kit (NEB) with 13 cycles used for PCR enrichment. AMPure XP beads were used for size selection and PCR purification. DNA yield was measured by Qubit 2.0 Fluorometer (Life technologies). For input and RFA1 libraries 34 μl from the RFA1 IP samples and 1 ng DNA in 34 ul water from the input was used as starting material. To generate complementary strand for the ssDNA first 5 μl 10 x NEB2.1 buffer and 5 μl of random primers (8N, 3 mg/ml stock) were added and the samples were boiled at 95°C for 5 minutes and immediately placed to ice for 5 minutes. 5 μl 10 x dNTP with dUTP instead of dTTP (2 mM each) and 1 μl T4 polymerase (NEB) were then added, and the mixture was incubated at 37°C in a thermal cycler for 20 min. 5 μl 0.5 M EDTA (pH 8) was immediately added to stop the reaction. This was then used to generate libraries using Ultra II library kit (NEB) with 16 cycles for PCR enrichment. Paired end sequencing was performed using NextSeq 500 (42 bp reads from each side) system. For experiments with normalization using S. Pombe cells, aliquots (~2.5 x 109 cells) of S. pombe cell pellets were resuspended in 250 μl SDS buffer, and 1/1000 volume of the original S. cerevisiae culture (corresponding to 1:10 S. pombe to S. cerevisiae ratio) was added to each S. cerevisiae samples which were then processed the same way as described above.