title: GSM5552165: Primed mESC H3K9me3 ICeChIP; Mus musculus; ChIP-Seq
library_construction_protocol: ICeChIP has been done as previously described88,92. Briefly, 10^7-10^8 plate adherent cells were released using Accutase (Millipore), quenched with complete medium and collected (500 rcf, 5 min., 4°C). Subsequent steps have been done on ice, cells after each wash were collected by centrifugation (500 rcf, 5 min., 4°C). Cells were washed twice with 10 ml PBS, twice with 5ml 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, 1x RL Protease Inhibitor Cocktail). Cells' membranes was lysed by adding 1 volume of the 2x Lysis Buffer (Buffer N supplemented with 0.6% NP-40 substitute (Sigma)) to the single cell suspension resuspended in 2 PCVs (packed cell volumes) of Buffer N. After 10 minutes incubation on ice, nuclei were collected by centrifugation and resuspended in at least 6 PNV (packed nuclei volumes) of buffer N. Subsequently, nuclei were layered over 7.5 ml Sucrose Cushion N (15 mM Tris pH 7.5, 15 mM NaCl, 60 mM KCl, 30% (w/v) Sucrose, 5 mM MgCl2, 1 mM CaCl2 1 mM DTT, 1x RL Protease Inhibitor Cocktail, 50 µg/mL BSA(NEB)) in a 50 ml centrifuge tube. Nuclei were spun through the sucrose cushion in swinging bucket rotor at 500 rcf for 12 min., 4°C. Nuclei were resuspended in 2 PNVs of buffer N, total nuclei acid content was measured spectroscopically at 260 nm by Nanodrop (Thermo Scientific) (1A260 = 50 ng/µl), prior to measurement, DNA was stripped from chromatin by adding 18 μl – 98 μl of 2 M NaCl and DNA was fragmented by vortexing and water bath sonication. The quality and quantity of nuclei was measured using a hemocytometer. The apparent concentration of chromatin was adjusted to 1 μg/μl with Buffer N. The following semi‑synthetic standards were then spiked in: symmetrical H3K4me3, H3K9me3, H3K27me3, H3K36me3, H3K79me2, H3K4me3-K27me3 cis‑bivalent, asymmetrical H3K4me3, H3K4me3-K27me3 trans-bivalent. To fragment the DNA, we aliquoted 100 µg of chromatin and added 1 Worthington unit of Micrococcal nuclease (Worthington) per 4.785 µg of chromatin (measured at 260nm, 1A260 = 50 ng/µl) and incubated at 37°C for 12 minutes. Digestion was stopped by adding 1/10 volume of 11x MNase stop buffer (110 mM EGTA, 110 mM EDTA pH 8.0). Subsequently, nuclei were lysed by slowly adding 5 M NaCl while mixing on a vortex (lowest setting) to the final concentration of 600 mM NaCl. Insoluble material has been spun down at 18000 rcf, 1min., 4°C. Hydroxyapatite (HAP) chromatography has been done as described in92. Briefly, 66 mg of HAP resin (Bio-Rad Macro-Prep® Ceramic Hydroxyapatite Type I 20 μm) was 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). Subsequently, 100 µg of digested soluble chromatin was added to the rehydrated resin and incubated for 10 minutes at 4°C on a rotator. Afterwards, resin slurry was transferred to the centrifugal filter unit (Millipore Ultrafree MC–HV Centrifugal Filter 0.45 μm). Resin was washed 4 times with 200 μl of HAP buffer 1, 4 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), and eluted 3 times with 50 µl HAP elution buffer (342 mM Na2HPO4 and 158 mM NaH2PO4 final pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 μM PMSF), each wash/elution step was accompanied by centrifugation step (600 rcf, 30 sec., 4°C). Concentration of the chromatin was evaluated spectroscopically by Nanodrop (Thermo Scientific) (1A260 = 50 ng/µl). Apparent concentration of the chromatin was adjusted to 20 ng/µl with ChIP buffer 1 with BSA (25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute, 100 µg/ml BSA(NEB)). ICeChIP was performed as previously described88 with following modifications: α-H3K4me3 ChIP- 5 µg chromatin, 2 µg 304M3B-1xHRV3C93, 40µl Streptavidin M-280 Dynabeads (Invitrogen). α-H3K9me3 ChIP- 3 µg chromatin, 0.5 µg 309M3B93, 10 µl Streptavidin M-280 Dynabeads (Invitrogen). α-H3K27me3 ChIP - 0.8 µg chromatin, 0.6 µg CST C36B11 lot 8, 5 µl Protein G Dynabeads (Invitrogen). Aforementioned volumes of magnetic beads were washed twice with 200 µl ChIP buffer 1 with BSA. Antibodies were resuspended in 100 µl of ChIP buffer 1 with BSA; subsequently, magnetic beads were collected using magnetic rack, supernatant was removed, and magnetic beads were resuspended with the antibody solution and incubated for at least 1 hr, 4°C, on a rotator. Afterwards unbound antibody was washed away with two 200 µl washes of ChIP buffer 1 with BSA. Streptavidin beads were additionally washed twice with 200 µl of ChIP buffer 1 with BSA supplemented with 5 µM biotin for 10 min, at 4°C, on a rotator for each wash, followed by wash with 200 µl ChIP buffer 1 with BSA. Supernatant was removed on a magnetic rack and specific amount of chromatin, mentioned at the beginning of this chapter, was used to resuspend the magnetic beads. Chromatin was incubated with antibody-beads conjugates for 10-15 minutes, at 4°C, on a rotator. Subsequently, magnetic beads were washed two times with 200 µl ChIP buffer 2 (25 mM Tris pH 7.5, 5 mM MgCl2, 300 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute, 100 µg/ml BSA(NEB)), and one time with 200 µl ChIP buffer 3 (10 mM Tris pH 7.5, 250 mM LiCl, 1 mM EDTA, 0.5% Na•Deoxycholate, 0.5%(v/v) NP 40 substitute, 100 µg/ml BSA(NEB)), 10 minutes, at 4°C, on a rotator with tube change after each wash. These washes were followed by quick 200 µl ChIP buffer 1 (without BSA) wash, and 200µl TE wash (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Chromatin was released from the resin using 50 µl ChIP elution buffer (50 mM Tris pH 7.5, 1 mM EDTA, 1% (w/v) SDS) at 55°C, for 5 minutes. Elution was supplemented with 200 mM NaCl, 10 mM EDTA, 10 µg of Proteinase K (Roche) and incubated at 55°C for 2 hours. DNA was isolated using 3 volumes of Serapure HD (1 mg/ml of 1 µm, hydrophobic, carboxylated, Sera-Mag SpeedBeads (GE 65152105050250), 20% PEG-8000, 2.5 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA, 0.05% Tween-20, filter sterilized prior to addition of magnetic beads), incubated for 5 minutes at room temperature, collected with magnetic rack and washed twice with >200 µl of 75% ethanol, on a magnetic rack without disturbing the magnetic beads. Subsequently, ethanol was carefully removed, and DNA was eluted by resuspending beads in 50 µl of TE buffer, magnetic beads were collected using magnetic rack and supernatant was moved to a new tube. In order to limit DNA loss, all operations have been performed using 250 µl siliconized tubes. reICeChIP was performed in the same manner as described above with following changes. For the primary IP we have used 5 µg of HAP purified chromatin, 2 µg 304M3B-1xHRV3C – HRV 3C cleavable, biotinylated, αH3K4me3 Fab (PDB:4YHZ)93, immobilized on 40 µl Streptavidin M-280 Dynabeads (Invitrogen). After 10 minutes incubation of chromatin with antibody-resin conjugate, resin was washed three times with 200 µl ChIP buffer 1 with 100 µg/ml BSA, each wash consisted of 10 minutes incubation at 4°C, on a rotator, followed by tube change. Subsequently, resin was briefly washed with 200 μl ChIP buffer 1 with 100µg/ml BSA, and chromatin was released from the resin with 20 µl of ChIP buffer 1 supplemented with 100 µg/ml BSA and 4 µg HRV3C incubated (GE Healthcare) on ice for 60 minutes, elution step was repeated one more time and both elutions were combined. HRV 3C endoprotease efficiently cleaves at its target sites at 4°C46 and has a wide range of chemical tolerance94, but it highly-specific for its cognate cleavage sequence95 permitting facile use in ChIP under conditions which preserve nucleosomes. Primary elution was added to 0.6 µg CST C36B11, αH3K27me3 mAb, immobilized on 5 µl of Protein G Dynabeads(Invitrogen) for 10 minutes, at 4°C, on a rotator. Subsequently, magnetic beads were washed two t