title: GSM3380972: WT Dox Hi-C^2, rep1; Mus musculus; Hi-C
library_construction_protocol: ChIP-seq: Harvested cells were crosslinked for 5 minutes in 1% formaldehyde.  Crosslinking was quenched by the addition of glycine to 125mM.  Fixed cells were PBS washed and cell pellets were frozen at -80C. Approximately 2x10^7 cells were used per ChIP. Pellets were resuspended in Lysis buffer 1 (25mM HEPES pH 7.5, 25mM EDTA, 0.5% SDS).  Chromatin was sheared in a Qsonica Q800R for 20 minutes at 40% power 30sec on 30sec off at 4C to ~150bp.  After centrifuging away debris, 4 volumes of Lysis buffer 2 (25mM HEPES pH 7.5, 187.5mM NaCl, 1.25% Triton X-100, 0.625% sodium deoxycholate, 6.25% glycerol, 12.5mM sodium pyrophosphate, 12.5mM B-glycerophosphate, 6.25mM NaF, 1.25mM Na3VO4) were added with protease inhibitors (1mM PMSF, 1ug/mL pepstatin, and 1 ug/mL leupeptin).  Sepharose 4B (Sigma-Aldrich) was washed with ChIP Wash Buffer 1 (25mM HEPES pH 7.5, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 5% glycerol, 5mM EDTA, 10mM sodium pyrophosphate, 10mM B-glycerophosphate, 5mM NaF, 1mM Na3VO4, protease inhibitors) and used to preclear the chromatin samples.  25μL/IP Protein G Dynabeads (ThermoFisher) were blocked in ChIP Wash Buffer 1 100ug/mL Acyclovir, 100ug/mL Zalcitabine, 5% Gly-Gly (Sigma-Aldrich). Beads were then washed with ChIP Wash Buffer 1.  Precleared chromatin, 2ug of anti-CTCF or anti-CTCF Ser224-P antibodies (Table 1), and blocked Protein G Dynabeads were incubated together overnight at 4C with rotation.  10% of the precleared chromatin input was also saved.  IP samples were washed with ChIP Wash Buffer 1, then with ChIP Wash Buffer 2 (25mM HEPES pH 7.5, 300mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 5% glycerol, 5mM EDTA, 10mM sodium pyrophosphate, 10mM B-glycerophosphate, 5mM NaF, 1mM Na3VO4, protease inhibitors) and then with TENG (50mM Tris-HCl pH 8.0, 1mM EDTA, 50mM NaCl, 5% glycerol, protease inhibitors).  IPs were incubated for 30minutes at 37C in TENG with 200ug/mL RNase A. 10% Input samples were also treated with 200ug/mL RNase A.  IPs were washed with ChIP Wash Buffer 2 and then TENG.  IPs were then eluted at 65C in Elution Buffer (50mM Tris-HCl pH7.5, 10mM EDTA, 1% SDS).  An equal volume of TE (Tris-HCl pH 8.0, 1mM EDTA) was added to the eluate with 40ug Proteinase K and incubated for 2hrs at 45C.   The input samples were similarly protease treated in parallel.  Both IP and input cross-links were reversed at 65C overnight and then phenol/chloroform/isoamyl alcohol extracted.  The aqueous phase was further chloroform extracted and then alcohol precipitated.  The DNA was suspended in TE. RNA-seq: RNA was purified by TRIzol (Thermo Fisher) extraction via manufacturer's instructions. Polyadenylated mRNA was the selected from total cell RNA using oligo(dT) beads (New England BioLabs). Hi-C^2: In-situ Hi-C was performed 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. HYbrid Capture Hi-C (Hi-C^2) probes were designed and hybridization to in-situ Hi-C libraries carried out as described previously (Sanborn et al., 2015). Probe sets were designed to enrich interactions in the region of interest: chrX:70,370,161-71,832,975 (mm9). Briefly, 120 bp probes were designed around the MboI restriction sites in the regions of interest and custom synthesized pools of single stranded oligodeoxynucleotides ordered from CustomArray, Inc. (Bothell, WA). Single stranded DNA oligos were amplified and biotinylated in a MAXIScript T7 transcription reaction (Ambion). The resulting biotinylated RNA probes were hybridized to 250-300 ng of in situ Hi-C libraries for 24 hours at 65C. DNA hybridized to the RNA probes was pulled down by streptavidin beads (Dynabeads MyOne Streptavidin C1, Life Technologies), washed, and eluted. Libraries were constructed using the NEBNext ChIP-seq Library Preparation Kit with no modifications to the protocol. We used 12-18 cycles of PCR amplification, depending on library concentration measured by qPCR.
identifiers.namespace: GEO
identifiers.id: GSM3380972