H2AK119ub (Cell Signaling Technology, 8240, lot 2)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
CHART-seq: Sheared chromatin from 2.5x10^7 cells was prepared as described earlier as in Simon et al., Nature 504, 465-469 (2013). 160 μL chromatin was mixed with 320 μL 2X hybridization buffer (50 mM Tris-HCl pH 7.0, 750 mM NaCl, 1% SDS, 1 mM EDTA, 15% formamide, 1 mM DTT, 200 mM PMSF, 1 cOmplete EDTA-free protease inhibitor cocktail tablet [Roche], and 100 U/mL RNase Inhibitor [Roche]) and pre-cleared with 60 μl MyOne Streptavidin C1 beads blocked with yeast total RNA and bovine serum albumin at room temperature for 1 hr. The pre-cleared chromatin was mixed with 36 pmol of either antisense (Xist-targeting) or sense (control) capture probes. We used a pool of 9 capture probes (biotinylated oligos). Hybridization was carried out at 37°C for 4 hr followed by incubation with 240 μL blocked MyOne Streptavidin C1 beads at 37°C for 1 hr. The beads were washed once with 1X hybridization buffer (1:2 mixture of sonication buffer and 2X hybridization buffer) at 37°C for 10 min, five times with wash buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 2% SDS, 2 mM EDTA, 2 mM EGTA, and 1 mM DTT) at 37°C for 5 min, and twice with elution buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 0.5% NP-40, 3 mM MgCl2, and 10 mM DTT) at 37°C for 5 min. CHART-enriched DNA was eluted by 20 μL RNase H (5 U/μL, New England BioLabs) in 200 μL elution buffer two times at 37°C for 30 min. Eluent was treated with 10 μL RNase A (20 mg/mL) at 37°C for 1 hr and then treated with SDS (final 1%), EDTA (final 10 mM), and 10 μL proteinase K (20 mg/mL) at 55°C for 1 hr. Reversal of crosslinks was performed by supplementing the eluent with NaCl (final 0.3 M) and incubation at 65°C overnight. DNA was extracted by phenol-chloroform and further sheared to below 500-bp fragments. ChIP-seq: Cells were crosslinked with 1% formaldeyde for 10 min, then nuclei were isolated and lysed. Chromatin was sheared by sonication to a size of ~200-400 bp. Cleared lysates were precleared for 1 hour, then 10% saved as input, and lysates were incubated with Dynabeads Protein G pre-bound with the antibody of choice. After stringent washes, ChIP DNA was eluted from beads, and together with input chromtain was reverse-crosslinked with proteinase K. DNA was purified by phenol/chloroform extraction and ethanol precipitation. RNA-seq: Total cell RNA was extracted using TRIzol (Thermo Fisher), from which mRNA was isolated using oligo(dT) beads and RNA-seq libraries prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs) as per manufacturer's instructions. in situ Hi-C: As described in Rao et al. (2014), 3-5 million cells were crosslinked with 1% formaldehyde for 10min. Crosslinked cell pellets were lysed with 300uL ice-cold HiC lysis buffer (10mM Tris HCl pH 8, 10mM NaCl, 0.2% Igepal CA630) with protease inhibitors (Roche). Cell suspension was incubated for 30min at 4C with rotation, then pelleted at 2500xg for 5min. Pelleted nuclei were then washed with 500ul ice-cold HiC lysis buffer and resuspended in 50uL 0.5% SDS solution and incubated at 62C for 10min. 145ul H2O and 25ul 10% Triton X-100 were added to quench SDS and nuclei incubated at 37C for 15min. 26.1ul of 10X NEB Buffer 2 and 100U of MboI restriction enzyme were added and nuclei digested overnight at 37C with rotation. MboI was then heat activated at 62C for 20min, then nuclei cooled to room temperature. To fill in MboI fragment overhangs and mark DNA ends with biotin, 50ul of fill-in master mix was added (37.5ul 0.4mM biotin-14-dATP, 1.5ul 10mM dCTP, 1.5ul 10mM dGTP, 1.5ul 10mM dTTP, 8ul 5U/ul DNA Polymerase I, Large (Klenow) Fragment) and mixed by pipetting. Nuclei were then incubated at 37C for 1h with rotation. To ligate biotinylated DNA ends together, 900ul ligation master mix was added (649ul H2O, 120ul 10X NEB T4 ligase buffer, 100ul 10% Triton X-100, 6ul of 20mg/ml BSA, 25ul of 400U/ul T4 DNA ligase). Reaction was mixed by inverting and incubated at room temperature for 4h with rotation. Reaction was then centrifuged at 1700xg for 5min at room temperature. Pelleted nuclei were resuspended in 750ul TES buffer (1mM EDTA, 10mM Tris-HCl pH 8, 0.5% SDS), 50ul Proteinase K (20mg/ml) added and incubated at 55C for 1h with rotation. To reverse crosslinks, 88.9ul 3M NaCl was added and crosslinks reversed by incubated overnight at 65C with shaking (Eppendorf thermomixer, 950rpm). Samples were cooled to room temperature and DNA extracted with phase-lock gel tubes (5Prime). RNA was digested from purifying DNA by adding 2ul RNAse A (20mg/ml) and incubating at 37C for 1h. DNA was then extracted with phase-lock gel tubes (5Prime) and resuspended in 130ul 1X Tris buffer (10mM Tris-HCl, pH 8). To ensure complete resuspension, DNA was then incubated at 37C for 15min. DNA was then sonicated to a size of 300-500bp using a Covaris with the following parameters: 140W, 10% duty cycle, 200 bursts/cycle, 100s. Samples were then size selected to 300-500bp using AMPure XP beads and eluted in 300ul 1X Tris buffer. To pulldown biotinylated ligation products, 30ul (per sample) of MyOne Streptavidin C1 beads were washed with 400ul of 1X Tween washing buffer (5mM Tris-HCl pH 7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween 20) and resuspended in 300ul 2X binding buffer (10mM Tris-HCl pH 7.5, 1mM EDTA, 2M NaCl). Beads were then added to purified DNA and sample incubated at room temperature for 15min with rotation. Beads were washed twice with 600ul 1X Tween Washing buffer. Subsequent library preparation steps were then done directly on beads. Libraries were constructed using the NEBNext ChIP-seq Library Preparation Kit with no modifications to the protocol. We used 9-14 cycles of PCR amplification, depending on library concentration measured by qPCR.