GSM6754610: NL_18_NPC_Input_ChIP; Homo sapiens; ChIP-Seq
ChIP-seq was performed as previously described with minor modification1-6. Briefly, crosslinked cell pellets (consisting of 10 million cells for CTCF ChIP-seq or 3 million cells for H3K9me3 ChIP-seq), were lysed in cell lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2 % NP-40/Igepal, Protease Inhibitor, PMSF) on ice for 10 min. The suspension was then homogenized with pestle A 30 times. The nuclei were pelleted from the initial lysate at 2,500 g at 4C and the resulting nuclei were further lysed in 500 μl of nuclear lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1 % SDS, Protease Inhibitor, PMSF) and incubated on ice for 20 min. Lysed nuclei were then sonicated by adding 300 μP IP Dilution Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 150 mM NaCl, 1 % Triton X-100, 0.01 % SDS, Protease Inhibitor, PMSF) and transferring to sonication tubes. Samples were sonicated using a QSonica Q800R2 sonicator for 1 hour set at 100% amplitude, with pulse set to 30 seconds on and 30 seconds off. The sonicated lysate was then pelleted at 14,000 RPM in 4 °C and the supernatant was transferred to a reaction consisting of 3.7 ml IP Dilution Buffer, 500 μl Nuclear Lysis Buffer, 175 μl of a 1:1 ratio of ProteinA:ProteinG bead slurry (Thermofisher, 15918014 and 15920010, respectively) and 50 μg of rabbit IgG for preclearing. The preclearing reactions were rotated at 4 °C for 2 hours. 200 ul of the pre-clearing reactions was saved as the “input” control. The remaining solution was added to an immunoprecipitation reaction consisting of 1 ml cold PBS, 20 μl Protein A, 20 μl Protein G, and 1 ul/million cells of either CTCF or H3K9me3 antibody and rotated overnight at 4 °C. The immunoprecipitation reactions were prepared one day before cell lysis and rotated overnight at 4 °C. The next day, IP reactions were pelleted and the supernatant was discarded. The remaining pellet was washed once with IP Wash Buffer 1 (20 mM Tris pH 8, 2 mM EDTA, 50 mM NaCl, 1 % Triton X-100, 0.1 % SDS), twice with High Salt Buffer (20 mM Tris pH 8, 2 mM EDTA, 500 mM NaCl, 1 % Triton X-100, 0.01% SDS), once with IP Wash Buffer 2 (10 mM Tris pH 8, 1 mM EDTA, 0.25 M LiCl, 1 % NP-40/Igepal, 1 % sodium deoxycholate) and twice with TE buffer (10 mM Tris pH 8, 1 mM EDTA pH 8). The IP DNA was eluted from the washed beads in Elution buffer (100 mM NaHCO3, 1 %SDS, prepared fresh) by resuspending and then spinning at 7,500 rpm. RNA was degraded with 2 ul RNAse A (Sigma, 10109142001)  and incubated at 65 °C for 1 hour. To degrade residual DNA, 3 ul proteinase K (NEB P8107S) was added and all samples were incubated overnight at 65 °C. DNA was extracted using phenol:chloroform and ethanol precipitation methods. Antibodies used in this study were: CTCF (Millipore 07-729), H3K9me3 (Abcam ab8898), IgG (Sigma I8140). ChIP-seq libraries were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit (NEB #7103) according to manufacturer's protocol. For ChIP-seq, size selection of adaptor-ligated libraries was performed using AgenCourt Ampure XP beads (Beckman Coulter, A63881) according to the manufacturer's protocol. For ChIP-seq, size selection targeted <1 kb fragment size and libraries were amplified using 11 PCR cycles. Input amounts for library preparation using the NEBNext Ultra II DNA Library Prep Kit were 1 ng of purified ChIP-seq libraries.