GSM1670908: MiSeq_PHH_H3K27ac; Homo sapiens; ChIP-Seq
Cells for chromatin immunoprecipitation (ChIP) were harvested with Accutase (Millipore) and crosslinked in PBS with 1% freshly cracked formaldehyde (Pierce) for 5 min. Fixation was quenched by adding 125mM glycine final concentration for 2 min. Next cells were washed with 10ml ice cold PBS with 0.1% Triton-X 100, 0.1% NP-40, 1mM DTT 10mM sodium butyrate, 1x protease inhibitor (Roche), resuspended in 1ml of the same buffer and incubated on ice for 10min with occasional mixing. Crude nuclei were pelleted and resuspended in Mnase digestion buffer (50mM Tris pH 7.5, 4mM MgCl2, 1mM CaCl2, 10% glycerol, 10mM sodium butyrate, 1x protease inhibitor) with 600U/ml Mnase (Fermentas). Chromatin was digested for 10 min at 37°C to mainly mononucleosomes before digestion was quenched with 10mM EGTA on ice for 30min. The digested nuclei were spun at 10,000 rpm for 5min and the supernatant (S1) saved into a fresh tube. The pellet was resuspended in Mnase buffer with 10mM EGTA and 300mM NaCl and mildly sonicated on ice 4x for 10 pulses in Heat Systems W-375 sonicator at 50% duty cycle and power setting 3. The sample was spun at 10,000 rpm for 5min and the supernatant (S2) combined with the S1 fraction. S1 and S2 fraction were mixed with an equal volume of 0% sucrose buffer (50mM Tris pH 7.5, 50mM NaCl, 5mM EDTA pH 8.0, 0.01% NP-40, 10mM sodium butyrate, 1x protease inhibitor) and concentrated in a Vivaspin 4 spin column (Sartorius) with 100K cutoff membrane to ~250ul. The concentrated sample was applied to a 5-30% sucrose gradient and centrifuged in a SW41TI at 40,000rpm at 4°C for 4h. Mononucleosome-containing fractions were pooled, concentrated to ~500ul and 100ng/ul BSA added. For ChIP antibodies were prebound to 20-30ul Protein G Dynabeads (Life Technologies) on a rotator at 4°C for 2h. The following antibodies were used: 0.5ug H3K4me3 (Abcam ab8580), 2ug H3K9me3 (Abcam ab8898), 1ug H3K27me3 (Millipore 07-449), 2ug H3K36me3 (Millipore ABE435), 0.6ug H3K27ac (Abcam ab4729), 1.2ug H3K122ac (Abcam ab33309), 2ug RNA Polymerase 2 (Abcam ab817). 1ug purified mononucleosomes (for histone modifications) or 20ug of Mnase-digested chromatin (for Polymerase 2 ChIP) were diluted in 400ul ChIP dilution buffer (20mM Tris pH 8.0, 150mM NaCl, 2mM EDTA pH 8.0, 1% Triton X-100, 10mM sodium butyrate, 1x protease inhibitor), added to the bead-antibody complex and incubated on a rotator at 4°C overnight. Samples were washed in LiCl was buffer (500mM LiCl, 50mM Tris pH 8.0, 1mM EDTA pH 8.0, 1% NP-40, 0.7% sodium deoxycholate) five times and eluted in elution buffer (1% SDS, 100mM NaHCO3). The samples were decrosslinked at 65°C for 5-6h in the presence of Proteinase K (Ambion) and purified with Qiagen PCR purification columns. ChIP DNA yield was quantified using QuBit Fluorometric Quantitation (Life Technologies). 1-2ng of DNA was used to prepare sequencing libraries with the KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer's instructions. For indexing, 2ul of adaptors from a TruSeq ChIP Sample Preparation Kit (Illumina) were used. Libraries were amplified with 13 PCR cycles and sequenced on a HiSeq2500 instrument (Illumina) in rapid run mode with 2x33bp paired end reads. Custom designed xGen Lockdown probes (Integrated DNA Technology) of 60bp each tiling the entire HBV genome with two-fold coverage were designed and equal amounts of libraries were pooled (500-1000ng DNA total amount) and enriched for HBV DNA according to the manufacturer's xGen rapid capture protocol v2 with slight modifications: Hybridization was performed at 50°C, and 50ul Dynabeads MyOne Streptavidin C1 (Life Technologies) were used per capture reaction. Target enriched libraries were amplified with 8 PCR cycles (KAPA HIFI HotStart ReadyMix) and sequenced on a MiSeq (Illumina) with 2x33bp paired end reads.
GEO Accession
GSM1670908