GSM8832481: SgD_tg_H3.3_rep1; Mus musculus; ChIP-Seq
Conventional ChIP: ChIP-seq in post-replicative cell populations (ScPD and rSt) was performed as follows. Chromatin from FACS-isolated ScPD and rSt populations was prepared by incubating cells for 20 min on ice in lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.2% sodium deoxycholate, 5 mM CaCl2) containing 5 U MNase (Roche Nuclease S7 10107921001) per 1M cells. After incubation on ice, chromatin was digested for 10 min at 37°C. The MNAse reaction was stopped by adding EGTA to 50 mM final concentration. The IP reactions were performed overnight at 4ºC on a rotator in the following proportions. To profile H3 isoforms, chromatin from 50K ScPD or 200K rSt was incubated with 5 µl of antibodies (anti-MYC: Abcam ab9132, anti-H3.3: Cosmo Bio CE-040B). To profile H3K4me3 and H3K27me3 in ScPD cells, chromatin from 1200K ScPD was incubated with 3 µl of antibodies (anti-H3K4me3: Millipore 17-614, anti-H3K27me3: Cell Signaling #9733). Per sample, 25 µl of 1:1 mix of protein A:protein G Dynabeads (Thermo Fisher Scientific, 10001D and 10003D) was washed once with 25 µl lysis dilution buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.2% sodium deoxycholate, 50 mM EGTA), then added to chromatin-antibody mix and incubated on a rotator for 4 h at 4ºC. The Chromatin-Antibody-Dynabeads complexes were washed twice with 200 µl of RIPA buffer (140 mM NaCl, 10 mM Tris-HCl (pH 8.0), 0.1% Sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 1 mM EDTA), once with 200 µl high salt buffer (140 mM NaCl, 10 mM Tris-HCl (pH 8.0), 0.1% Sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 1 mM EDTA), once with 200 µl LiCl wash buffer (250 mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, (pH 8.0)), and once with 200 µl TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Finally, chromatin was eluted and digested for 1 h at 63ºC in Elution buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% SDS, 300 mM NaCl). DNA from eluted material was purified by 2X volumes of Ampure XP DNA purification beads (Beckman Coulter, A63881) according to manufacturer's instructions and resuspended in 9 µl of nuclease-free water. . ULI-ChIP: To profile the H3 isoforms, chromatin from 5K SgU or 10K SgD, ScLZ or ScPD cells were used with 0.5 µl of anti-H3.3 (Cosmo Bio CE-040B) or 0.5 µl anti-Myc (Abcam ab9132) antibodies per sample. ULI-ChIP-seq libraries were prepared from the eluted ChIP DNA using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645L) Conventional ChIP: The eluted ChIP DNA was used to prepare libraries using the NEB Ultra protocol according to the manufacturer's instructions. The libraries were sequenced in a 50 pb single end mode on the Illumina HiSeq 2500 platform, according to the manufacturer's instructions. ULI-ChIP: Libraries were sequenced in 50-bp paired-end mode with Illumina NovaSeq 6000 using the manufacturer's instructions