GSM3729101: ChIP-seq-CTCF-prometa-rep1; Mus musculus; ChIP-Seq
For each IP, 7-10million crosslinked cells were re-suspended in 1ml Cell Lysis Buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40/Igepal) containing fresh protease inhibitors (Sigma P8340) and 1mM phenylmethylsulfonyl fluoride (PMSF) for 20min on ice. Nuclei was pelleted at 4℃ and re-suspended in 1ml Nuclear Lysis Buffer (50mM Tris pH 8, 10mM EDTA, 1% SDS) containing fresh protease inhibitors and PMSF and incubated for 20min on ice. Samples were added with 0.6ml IP Dilution Buffer (20mM Tris pH 8, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS) containing fresh protease inhibitor and PMSF and then subject to sonication for 45min (Epishear, Active Motif, 100% amplitude, 30s ON and 30s OFF, ~45min). After sonication, samples were spun at 21130g at 4℃ for 10min to pellet cell debris. Supernatant was transferred to a new tube and supplemented with 3.4ml IP Dilution Buffer containing fresh protease inhibitor, PMSF, protein A/G agarose beads (prepared by mixing protein A (Thermo Fisher Scientific 15918014) and protein G (Thermo Fisher Scientific 15920010) agarose beads at 1:1 ratio) and 50mg isotope matched IgG. Chromatin preclear was carried out for over 2h at 4℃. 200ml (4%) of precleared chromatin was taken out from subsequent IP steps and set as input. Pre-cleared chromatin was then added to protein A/C beads bound with antibodies and incubated overnight with rotation at 4℃. After IP, beads were washed once with IP Wash Buffer I (20mM Tris pH 8, 2mM EDTA, 50mMNaCl, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20mMTris pH 8, 2mM EDTA, 500mMNaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash Buffer II (10mMTris pH 8, 1mM EDTA, 0.25 M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate) and twice with PE Buffer (10mM Tris pH 8, 1mM EDTA pH 8). All washing and pelleting steps were performed on ice or at 4℃. Beads were then moved to room temperature and eluted twice with 100ml freshly prepared Elution Buffer (100mM NaHCO3, 1%SDS) to reach a final concentration of 200ml. Eluted samples were supplemented with 12ml 5M NaCl, 2ml DNase-free RNaseA (10mg/ml, 10109169001 BMB) and incubated at 65℃ for overnight. Samples were then added with 3ml protease K (20mg/ml, 3115879 BMB) and further de-crosslinked at 65℃ for 2h. De-crosslinked DNA was added with 10ml 3M sodium acetate pH 5.2 and purified with QIAquick PCR Purification kit (QIAGEN 28106) according to manufacture instruction. Library construction was performed using Illumina's TruSeq ChIP sample preparation kit (Illumina, catalog no. IP-202-1012) according to manufacturer's specifications with the addition of size selection (left side at 0.9x, right side at 0.6x) using SPRIselect beads (Beckman Coulter, catalog no. B23318) prior to PCR amplification. Library size was determined (average 351 bp, range 333-372 bp) using the Agilent Bioanalyzer 2100, followed by quantitation using real-time PCR using the KAPA Library Quant Kit for Illumina (KAPA Biosystems catalog no. KK4835). Libraries were then pooled and sequenced on the Illumina NextSeq 500 platform using Illumina sequencing reagents according to manufacturer's instructions