GSM8521310: Biol rep 4 TMH3K27 A366 treated; Betapolyomavirus macacae; ChIP-Seq
Chromatin immunoprecipitation (ChIP) analyses of the SV40 chromosomes obtained by treatment with DMSO, or inhibitor were performed according to the detailed description of the procedures previously described (Balakrishnan and Milavetz, 2017b;a). All the antibodies used were ChIP validated by their respective vendors. Antibodies (and their vendors) included: acetyl-H3 (06-599, Millipore), acetyl-H4 (06-866, Millipore), H3K9me1 (ab9045, Abcam), H3K27me3 (17-622 Millipore), and RNAPII (05-623-25G, Millipore). ChIPs were performed using Millipore kit Cat. No. 17-295 according to the supplier's protocol. In a standard ChIP, 10 µl of antibody (10 µg) was incubated with the SV40 chromosomes being analyzed for 4 hours at 40C. A mixture of magnetic beads containing protein A and protein G was added (15 µl each) to each sample and incubated overnight. The next morning the magnetic beads and associated antibody bound chromatin was purified according to the protocol present in the kit. The remaining DNA sample 25 µl was used for the preparation of a sequencing library. Libraries were prepared using the New England Biolabs Next UltraII kit according to the protocol supplied with the kit with one exception. All reagent volumes were reduced by half for the preparation of libraries with no apparent effect on library quality or quantity. Following its preparation, the library was purified AMPure magnetic beads in a two-step process. First, 7.5 µl of AMPure was added to the library (50 µl) in a 1.4 ml Eppindorf tube and incubated for 10 minutes to bind the DNA. The tube was incubated for 5 minutes in a magnetic rack and the liquid transferred to a new Eppendorf tube. An additional 20 µl of AMPure was added and incubated for 10 minutes. The tube was again placed on the magnetic rack and after a 5-minute incubation the liquid was removed and discarded. The magnetic beads were washed twice with 400 µl 70% ethanol, dried for 10 minutes, and resuspended in 20 µl nuclease-free water. In preliminary studies we found that this two-step purification yielded libraries enriched for nucleosome-sized insert DNA while also removing residual adapter from the library preparation. The size-selected library from each sample, 5 µl, was amplified by PCR using indices from New England Biolabs and purified on AMPure as previously described with minor modification (Balakrishnan and Milavetz, 2017a). Following PCR the amplification products, 170 µl were again purified in a two-step AMPure procedure. In the first step 60 µl of AMPure was added, the beads bound to a magnetic rack and the liquid transferred to a new tube. AMPure, 40 µl, was added to the liquid and the purification continued as previously described. This two-step process yielded a final sequencing library that was primarily in the size range of 150 to 300 base pairs in length including the adapter sequences from the library preparation. ChIP samples for library preparation for ChIP-Seq was performed as above until the final elution step. Instead of eluting the intact SV40 chromatin bound to magnetic beads, the chromatin bound to beads was sonicated to fragment the chromatin and the fragments bound to the beads separated from the unbound fragments. The beads were washed twice with TE buffer from the Millipore kit. The DNA in the chromatin fragments still bound to the magnetic beads was removed by incubation for 15 minutes in the DNA binding buffer found in the NEB Monarch PCR and DNA Cleanup Kit. The DNA present in binding buffer was then purified according to the protocol found in the Monarch kit. In the final purification step with the kit, the DNA was eluted in 26 μl nuclease-free H2O. In order to estimate the amount of SV40 DNA present in the eluate, 1 µl was amplified along with a 1 µl sample of the SV40 chromatin used in the original ChIP using the early primers.