Anti ctcf antibody

ChIP was carried out following standard protocols. Briefly, 4-million cells were fixed with 1% formaldehyde for 10 min and sonicated on ice at 40% amplitude (30 s on, 90 s off, for a total of 24 min) using a Branson 101-135-126 Sonifier. ChIP was performed using 5 μl of a polyclonal anti-CTCF antibody (Millipore 07–729), 10 μl monoclonal anti-CSB antibody (1B1) (9 (link)) and 5 μl recombinant protein-G agarose beads (Invitrogen). ChIPed DNA was analyzed by real-time PCR using a 7900HT Fast Real-Time PCR System from Applied Biosystems and SYBR green. Primers were as described in Supplementary Table S7. For all ChIP-qPCR experiments described in this manuscript, menadione treated and untreated cells were examined side-by-side. For the ChIP-seq experiments, the CSB+M sample was processed alongside one untreated sample, which was previously reported (9 (link)).
For western blot analyses, ChIP samples were reverse cross-linked in SDS sample buffer at 95°C for 30 min (8 (link)).

The endogenous protein levels of CTCF were observed by SDS-PAGE/western blot analysis [31 (link),32 (link)] in whole cell lysates of 226LDM cells from the control and treated populations using a polyclonal anti-CTCF antibody (Millipore, 07–729, lot # JBC1903613, pre-screened with the lysates from breast normal and tumour tissues to detect both, CTCF130 and CTCF180). Anti-tubulin specific antibody (SIGMA, T5168) was used as a loading control. Chemiluminescence detection was performed with the Fusion FX7 gel documentation system (PeqLab) and the UptiLight (Interchim) reagents according to the manufacturer's instructions.

ChIP was performed as described previously (Lee et al., 2006 (link)). Approximately 30 million cells were crosslinked for 10 min at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution to the growth media followed by 5 min quenching with 125 mM glycine. Cells were washed twice with PBS, and then the supernatant was aspirated and the cell pellet was flash frozen at −80°C. 100 µL Protein G Dynabeads (Thermo 10003D) were blocked with 0.5%BSA (w/v) in PBS. Magnetic beads were bound with 40 µL anti-CTCF antibody (Millipore 07–729). Nuclei were isolated as previously described (Lee et al., 2006 (link)) and sonicated in lysis buffer on a Misonix 3000 sonicator for 5 cycles at 30 s each on ice (18–21 W) with 60 s on ice between cycles. Sonicated lysates were cleared once by centrifugation and incubated overnight at 4°C with magnetic beads bound with antibody to enrich for DNA fragments bound by the indicated factor. Beads were washed with wash buffers A, B, C, and D sequentially. DNA was eluted, cross-links were reversed, and DNA was purified with phenol chloroform extraction and ethanol precipitation. Libraries for Illumina sequencing were prepared following the Illumina TruSeq DNA Sample Preparation v2 kit and sequenced on the Illumina HiSeq 2500 for 40 bases in single-read mode.

After post-sort to confirm the sorted population and to count cell numbers, HSPCs (lineage- c-kit + ) were washed with PBS once and then lysed in 1 ml IP buffer (50 mM Tris-Cl pH 8.0, 100 mM Na Fluoride, 30 mM Na Pyrophosphate, 2 mM Na Molybdate, 5 mM EDTA, 2 mM Na Vanadate, 1% NP-40, and freshly prepared protease inhibitor purchased from Merk, cat.no. 11697498001) on ice for 10 min with occasionally inverting. After spinning down at 13,200 rpm for 10 min at 4 °C, the supernatant was transferred into a new tube containing antibody pre-bound dynabeads protein beads for overnight incubation at 4 °C (anti-CTCF antibody, #07-729, Millipore; Anti-ZNF143 rabbit serum is generated in our lab); 50 µl of the supernatant was stored at -20 °C as input. The next day, after washing beads for five times with IP buffer, loading dye was added to the beads and input for boiling at 97 °C for 10 min, followed by Western-blot analysis.

ChIP samples were prepared as described [69 (link)]. Briefly, chromatin from GFP or Oct4 KD ESCs was crosslinked, lysed and sonicated to generate 300-1000 base-pair fragments. 50 μl of Protein A Magnetic beads (NEB) were washed twice with PBS containing 5 mg/ml BSA and 10 μl of anti-CTCF antibody (Millipore) was coupled in 500 μl PBS with 5 mg/ml BSA overnight at 4°C. Immunoprecipitation was performed with antibody-coupled beads and sonicated supernatants in ChIP buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) overnight at 4°C. Magnetic beads were washed twice with ChIP buffer, once with ChIP buffer including 500 mM NaCl, 4 times with RIPA buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na⋅Deoxycholate), and once with TE buffer (pH 8.0). Chromatin was eluted twice from washed beads by adding elution buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS) and incubating for 15 minutes at 65°C. Crosslinking was reversed at 65°C for 6 hr and RNase A/T1 (Ambion) was added for 1 hr at 37°C followed by proteinase K (Ambion) treatment overnight at 50°C. ChIP-enriched DNA was purified using Phenol/Chloroform/Isoamyl alcohol extractions in phase-lock tubes. Then, chromatin was analyzed by qPCR as described above, using primers specific for CTCF sites of interest (Additional file 5).