ZFP14 ChIP input
Cell culture and transfection. 293T cells were cultured in DMEM medium supplemented with 1% of Penicillin G and 10% FBS and maintained at 37。 in a humidified incubator. The ORF of each of the ZFPs: ZFP1, ZFP14, ZNF682 or ZNF135 was cloned into the pLenti6/V5-DEST vector that allows expression of V5 epitope c-terminal tagged protein. The cells were plated at the density of 3*104 per cm2 and transfected with one of the three plasmids or with an empty vector using FuGENE 6 reagent (Promega) 24h later. The cells were PFA fixed and collected 48h after transfection. The protein expression was verified by western blot using an anti-V5 antibody. 2. Preparation of Chromatin: Cells we fixed with 1% of PFA for 10 minutes, followed by addition of glycine (0.125M final concentration). Cross-linked cells were washed twice with ice cold PBS, resuspended in swelling buffer (25mM HEPES pH7.8, 1.5mM MgCl2, 10mM KCl, 0.1% NP40, 1mM DTT and PIC (protease inhibitors cocktail, Roche)) and incubated on ice for 10 minutes. Nuclei were collected by the centrifugation, washed once with swelling buffer and the pellets were re-suspended in sonication buffer (20mM Tris-HCl pH=8.0, 150mM NaCl, 2mM EDTA, 1% TritonX-100, 0,1% Na Deoxycholate, 1% SDS, 1mM DTT and PIC) and sonicated using Covaris to obtain the fragments of 200-300 bp. The chromatin was centrifuged at 14,000 rpm to remove the debris and an aliquot of 50無 was taken as an input. To reverse cross-link the samples, 20無 of 5M NaCl, 1無 of RNaseA (10痢/mL) and water were added to a final volume of 500無. The samples were then incubated overnight at 65。 and treated with 0.05mg/mL proteinase K at 45。 for 1 hour. DNA was purified with a PCR purification kit (Qiagen). The sonication efficiency and the size of the fragments were verified using agarose gel electrophoresis and the DNA concentration was measured with Nanodrop (Thermo Fisher). 3.IP: For each immunoprecipitation 25痢 of pre-cleared chromatin was incubated with either 20無 of Anti-V5 Agarose Affinity Gel (Sigma-Aldrich) or protein-G agarose beads (Roche) at 4。 overnight. After that beads were washed twice with each of the following buffers at 4。: 1mL of Low Salt (20mM Tris-HCl pH=8.0, 150mM NaCl, 2mM EDTA, 1% TritonX-100 and 0.1% SDS), 1 mL of High Salt (20mM Tris-HCl pH=8.0, 500mM NaCl, 2mM EDTA, 1% TritonX-100 and 0.1% SDS), 1mL of LiCl (10mM Tris-HCl pH=8.0, 250mM LiCl, 1mM EDTA, 1% NP40 and 1% Na Deoxycholate) and 1 mL of TE (10mM Tris-HCl pH=8.0 and 1mM EDTA). Beads were incubated in the elution buffer (1% SDS, 0.1M NaHCO3) for 15 min at room temperature and the supernatants were processed for the reverse cross-link as described previously. 4. Library preparation: Libraries were prepared as described in46 except for the size selection step, which was substituted with a double-size selection by AMPure XP beads (with a 100bp average insert size) (NEB E6270).