GSM1565719: H3K36me3_ICeChIP-seq_mESC-E14; Mus musculus; ChIP-Seq
Our ICeChIP protocol is based on input preparation largely following the Dilworth lab native ChIP protocol (Brand et al., 2008). Plate-adhered cells  (~107 cells) were washed twice with 10 mL of PBS, and released by 5 mL Accutase (Millipore) for 5 minutes in 37°C, quenched with 2 mL of complete media, and collected by centrifugation (500 x g, for 5 minutes at 4°C). All subsequent steps were performed on ice with ice-cold buffers. Cells were washed twice with 10 mL PBS, and twice with 5 mL of Buffer N (15 mM Tris pH 7.5, 15 mM NaCl, 60 mM KCl, 8.5% (w/v) Sucrose, 5 mM MgCl2, 1 mM CaCl2 1 mM DTT, 200 μM PMSF, 1x RL Protease Inhibitor Cocktail). Cells were resuspended in 1mL of Buffer N and lysed by adding 1 mL of 2x Lysis Buffer (Buffer N supplemented with 0.6% NP-40 substitute (Sigma)) for 10 minutes at 4°C. Nuclei were collected by centrifugation (500 x g for 5 minutes at 4°C) and were resuspended in 6 PCVs (packed cell volumes) of Buffer N. To remove cell debris, resuspended nuclei were overlaid on the surface of 7.5 mL of sucrose cushion (10 mM HEPES pH 7.9, 30%(w/v) sucrose, 1.5 mM MgCl2) in a 50 mL centrifuge tube centrifuged (1300 x g, Sorvall Legend XTR swinging bucket rotor for 12 minutes at 4°C). Most cell debris remained in upper layer while nuclei sedimented through the sucrose cushion and pelleted on the bottom of the tube.  The supernatant was discarded and nuclei were resuspended in 2 PCVs of Buffer N. To measure apparent concentration of chromatin, 2 μL of resuspended nuclei were diluted in 98 μL of 2M NaCl in triplicate, total nucleic acid absorbance was measured at 260 nm by Nanodrop (Thermo Scientific), and the conversion factor assuming 1A260 = 50 ng/μL of chromatin employed. Based on these measurements, apparent concentration of chromatin was adjusted to 1 μg/μL with Buffer N. The quantity and quality of nuclei were also assessed using hemocytometer. Chromatin was sheared through exhaustive MNase digest.  At this stage, a ladder of semisynthetic nucleosomes was doped into the pool of native nucleosomes.  The amount of spiked ladder was comparable to estimated amount of genome copies in the pool based on the nuclei counting times the average DNA content per cell (~2.5 copy of genome per cell).  To remove debris coming from nuclei lysis and MNase digestion as well as strip chromatin bound factors, the pool of nucleosomes was subjected to hydroxyapatite chromatography purification (Brand et al., 2008).  Fragmented chromatin with internal standard ladders were split into 100 μg total nucleic acid fractions and each fraction was mixed with 66 mg of hydroxyapatite (HAP) resin (Bio-Rad Macro-Prep® Ceramic Hydroxyapatite Type I 20 μm) rehydrated with 200 μL of HAP buffer 1(3.42 mM Na2HPO4 and 1.58 mM NaH2PO4 final pH 7.2, 600 mM NaCl, 1 mM EDTA, 200 μM PMSF), incubated for 10 minutes at 4°C on rotator and subsequently was applied to the centrifugal filter unit (Millipore Ultrafree MC–HV Centrifugal Filter 0.45 μm). The chromatin-loaded resin in the column was drained and then washed four times with 200 μL HAP buffer 1 and four times with 200 μL of HAP buffer 2 (3.42 mM Na2HPO4 and 1.58 mM NaH2PO4 final pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 μM PMSF) by centrifugation (600 x g, 1 minute at 4°C in fixed angle rotor. Nucleosomes were eluted from the HAP column with three 100 μL washes of HAP elution buffer (342 mM Na2HPO4 and 158 mM NaH2PO4 final pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 μM PMSF). To measure apparent concentration of HAP purified chromatin fragments, 10 μL of HAP elution was diluted in 40 μL of 2M NaCl in triplicate, and absorbance measured at 260 nm averaged and adjusted (1A260 = 50 ng/μL of chromatin). Apparent concentration of chromatin was adjusted to 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute). H3K4me3 ChIP was performed with 10 μg of chromatin and 15 μL of AM39159 antibody, H3 and H4 ChIP was performed with 1 μg of chromatin and 15 μL of AM61277 and AM61299 antibody, respectively (Active Motif). 10% of initial chromatin for each IP was set aside to serve as ChIP input. Each IP experiment used 50 μL of Protein A Dynabeads (Invitrogen) that were washed twice with 1mL of ChIP buffer 1 with 1 min collection on magnetic rack after each wash. To prepare the resin, 15 μL of antibody and 85 μL of ChIP buffer 1 was added to Protein A Dynabeads and incubated for 10 minutes at room temperature on a rotator, then washed twice with 1mL of ChIP Buffer 1. Chromatin (10 μg unless otherwise indicated) in 500 μL of ChIP buffer 1 was then added to magnetic beads and incubated for 15 minutes at room temperature on rotator. Beads were washed 3 times with 1 mL of ChIP Buffer 2 (mM Tris pH 7.5, 5 mM MgCl2, 300 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute), then twice with ChIP buffer 3 (10 mM Tris pH 7.5, 250 mM LiCl, 1mM EDTA, 0.5% Na•Deoxycholate, 0.5%(v/v) NP 40 substitute), each wash consisting of a 10 minute rotating incubation and 1 minute collection on magnetic rack at 4ºC.  During the course of washing, at least two tube changes reduced non-specific background.  Beads were then rinsed with 1 mL of ChIP Buffer 1 and 1 mL of TE buffer, followed with two 200 μL ChIP elution buffer steps (50 mM Tris pH 7.5, 1 mM EDTA, 1% w/v SDS). Each elution step consisted of 10 minute incubation at 65ºC in a Thermoshaker (Eppendorf) at 900 rpm. Elutions were combined and ChIP elution buffer was added to inputs to match volume of ChIP elutions. After adjusting the buffer to 200mM NaCl, 100 ng of RNase A was added into the mixture and incubated at 65ºC for 45 minutes in Thermoshaker at 800 rpm, and terminated with 10 mM EDTA. Next, protein digestion was accomplished with 20 ug of proteinase K (Roche) for 2 hrs at 42ºC in the Thermoshaker at 800 rpm. DNA was recovered and purified with Qiaquick columns (Qiagen): 6 volumes of PB buffer were added to the digestion and this solution applied to the column (17900 x g, 30s) followed by 3x 750 μL of PE buffer washes (17900 x g, 30s) with an extra 1 minute spin to remove residual ethanol. DNA was eluted by applying two times 25 μL of TE buffer at 50ºC and centrifuging (17900 x g, 1 min). For library preparation 10 ng of DNA isolated from IP or input was used. In cases in which the total amount of DNA was below 10 ng, all available DNA was subjected to library preparation.  Ends of DNA were blunted using the End-it™DNA End-Repair Kit (Epicentre) (7μL 10x End-It buffer, 7μL 2.5 mM dNTP Mix, 7μL 10 mM ATP, 1.4μL of  End-Repair Enzyme Mix and 47.6μL of DNA in TE buffer, incubated for 45 minutes at room temperature. DNA was purified with 126μL (1.8 volume) of Ampure XP Beads (Beckman Coulter). Beads were mixed with End repair mixture by pipetting 10 times up and down followed by 5 minutes incubation at room temperature. Magnetic beads were collected on side of the tube by magnet and two 30 sec 250 μL 80% EtOH washes on magnet were performed. Tubes were removed from the magnetic rack and 34 μL of TE buffer was added to beads and pipetted 10 times up and down. Magnetic beads were not removed from elution and remained in the tube during A-tailing. Addition of single adenosine to 3’ ends of DNA was accomplished by adding to 5 μL NEB buffer 2, 10 μL 1 mM dATP, 1 μL Klenow fragment (3’→5’ exo-, NEB) to the End-repaired DNA, and with incubation at 37ºC for 30 minutes. To purify DNA, 110 μL (2.2 volume) of SPRI Buffer (20% PEG6000, 2.5M NaCl) was added to the reaction and was pipetted 10 times up and down followed by 5 minutes incubation at room temperature. Magnetic beads were collected on side of the tube with magnet and two 30 sec 200 μL 80% EtOH washes on magnet were performed. Tubes were then taken out of magnetic rack and 13 μL of TE buffer was added to beads and mixed by pipette. Magnetic beads were not removed from elution and remained in the tube during adaptor liga