yH2A (1.6 ug/ml final concentration, Abcam, ab181447)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Pellets from 50 ml culture were resuspended in 500 μl SDS buffer (1% SDS, 10 mM EDTA, 5M Tris HCl, cOmplete Tablets, Mini EDTA-free EASYpack (Roche), PhosSTOP (Roche)). Cells were lysed in a FASTPREP machine, 5 rounds of 1 min at 6.5 power, with 200 μl of 0.5 mm silica beads. Lysate was spun out 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 a final volume of 1 ml. Samples were sonicated using the Focused-Ultrasonicator (Covaris, M220) (Average incident power – 7.5 Watts, Peak Incident Power – 75 Watts, Duty Factor – 10 %, Cycles/Burst – 200, Duration – 20 min). The sample was centrifuged for 20 min at 13,000 rpm at 4°C. Supernatant was then diluted to 1:10 (5 ml total). 50 μl protein A Dynabeads (Invitrogen, 10002D) and 50 μl protein G Dynabeads (Invitrogen, 10004D), were washed 3 times in IP buffer followed by adding to the sample and incubating for 2 h at 4°C. Supernatant was split, with 2X 2 ml being taken to 15 ml Falcon tubes, and 1 ml being kept at -20°C as an input sample. To the two 2 ml samples antibody was added, either H2A 1:500 (active motif) or 1.6 μg/ml H2AP (Abcam), and these were placed on a rotating wheel at 4°C for 15 – 20 h. For experiments where RPA1 ChIP was performed on the same sample, 75 ml starting cultures were used meaning supernatant could be split into 3X 2ml and 1 ml for input, with RPA1 antibody (1:10000, Agrisera) added to one 2 ml aliquot. For experiments where RPA1 ChIP was exclusively performed, 25 ml starting cultures were used to make a split of 1X 2ml for antibody addition and 1ml for input. A preparation of Dynabeads (Invitrogen), Protein A (30 μl) and Protein G (30 μl), was washed 3 times in IP buffer. This was added to each sample and incubated at 4°C for 4 h. Supernatant was removed and beads were 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). Elution was carried out in 400 μl elution buffer, for 30 min at room temperature. At the same time 50 μl from the input sample was added to 150 μl of elution buffer. 20 μl of 5 M NaCl and 10 μl of 10 mg/ml proteinase K (Invitrogen) was then added to the input, and 40 μl and 20 μl to the IP samples respectively. These were incubated at 65°C overnight. Then 10 μl of DNase-free RNase (Roche) was added to the input and 20 μl to the IP samples, and they were left at 37°C for 30 min. All DNA was purified with a Qiagen PCR purification kit and eluted in 50 μl for H2A or H2AP or 40 μl for RPA1. DNA amount was measured using the Qubit 2.0 Fluorometer (Life technologies) as per the manufacturer's instructions. For H2A or H2AP samples, libraries were prepared using the NEBnext Ultra II library kit (NEB) as per the manufacturer's instructions. PCR enrichment required 13 cycles. PCR purification was carried out using AMPure XP beads. For RPA1 library preparation 34 ul from the RPA1 samples and 1 ng DNA in 34 ul water from the input were used. 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 added and the mixture was incubated at 37°C in a thermal cycler for 20 min, and 5 µl 0.5 M EDTA (pH 8) was immediately added to stop the reaction. The resulting dsDNA was used to create libraries using the Ultra II library kit (NEB) as per the manufacturer's instructions.