GSM2773397: K562_AB-7766_H3K4me2_ICeChIP; Homo sapiens; ChIP-Seq
K562 cell pellets were washed twice with 5 mL of PBS, then washed twice with 5 mL of Buffer N (15 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, 8.5% w/v Sucrose, 5 mM MgCl2, 1 mM CaCl2, 1 mM DTT, 200 uM PMSF, 50 ug/mL BSA, 1x Roche Protease Inhibitor Cocktail, Filter Sterilized), with each wash consisting of complete resuspension of the pellet, centrifugation at 500 g for 5 minutes at 4°C, and removal of supernatant. The washed pellet was then resuspended in at least 2 packed cell volumes (PCV) of Buffer N and mixed with 1 volume of Lysis Buffer (Buffer N supplemented with 0.6% NP-40 Substitute) and incubated on ice for 10 minutes to lyse cells. The crude nuclei were spun down at 500 g for 5 minutes at 4°C before being resuspended in at least 6 packed nuclear volumes (PNV) of Buffer N and applied to the top of 7.5 mL of Sucrose Cushion N (15 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, 30% w/v Sucrose, 5 mM MgCl2, 1 mM CaCl2, 1 mM DTT, 200 uM PMSF, 50 ug/mL BSA, 1x Roche Protease Inhibitor Cocktail, Filter Sterilized) in a 15 mL centrifuge tube, then spun down at 500 g for 12 minutes at 4°C in a swinging-bucket rotor. The supernatant was discarded after centrifugation, and the pellet was resuspended in approximately 2 PNV of Buffer N. The nuclei were then quantified by diluting 2 uL of nuclei suspension into 48 uL of 2 M NaCl, water-bath sonicating to solubilize DNA, and spectroscopically measuring DNA concentration by Nanodrop measurement, where 1 A280nm¬ = 50 ng/uL chromatin in the measurement sample. After accounting for the 25-fold dilution of the measurement sample, the concentration of the nuclei was adjusted down to 1ug/uL of chromatin. Nuclei were aliquoted into 100 uL aliquots, flash frozen, and stored at -80°C until use. When nuclei aliquots were used, they were first thawed and spiked approximately 1 uL of each barcoded nucleosome standard per 50 ug of chromatin to be used. The nuclei suspension with spiked calibrants was then mixed with a pipette and transferred to a new tube, then warmed to 37°C for 2 minutes. After warming, 1 unit of micrococcal nuclease (MNase) per 4.375 ug of chromatin was added to the nuclei suspension and incubated at 37°C while shaking at 900 rpm for 12 minutes. After incubation, the digestion was stopped by adding 1/9 volume of 10x MNase Stop Buffer (100 mM EDTA, 100 mM EGTA, Filter Sterilized) while slowly vortexing, and nuclei were lysed by adding 5 M NaCl to a final concentration of 600 mM NaCl while slowly vortexing. 66 mg of HAP resin (BioRad, CHTTM Ceramic Hydroxyapatite, Type I, 20 um) per 100 ug of chromatin digested was rehydrated with 200 uL of HAP Buffer 1 (5 mM Sodium Phosphate pH 7.2, 600 mM NaCl, 1 mM EDTA, 200 uM PMSF, Filter Sterilized) per 100 ug of chromatin digested. The lysed nuclei were then spun down at 18,000 g for 1 minute to pellet insoluble nuclear debris, and the soluble fraction was added to the rehydrated HAP resin and incubated for 10 minutes on a rotator at 4°C. After incubation, the HAP resin slurry was added to a centrifugal filter unit (Millipore Ultrafree MC–HV Centrifugal Filter 0.45 um) and spun at 1000 g for 30 seconds at 4°C. The HAP resin left on the filter unit was then washed 4 times with 200 uL HAP Buffer 1 and 4 times with 200 uL HAP Buffer 2 (5 mM Sodium Phosphate pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 uM PMSF, Filter Sterilized) by spinning at 1000 g for 30 seconds at 4°C. Into a clean tube, the HAP resin was eluted with three elutions of 100 uL HAP Elution Buffer (500 mM Sodium Phosphate pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 uM PMSF, Filter Sterilized). The DNA content of the elution was then quantified spectroscopically with a Nanodrop, and the chromatin concentration was adjusted to 20 ng/uL 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, Filter Sterilized) with 100 ug/mL of BSA. With the exception of the Koide Lab 304M3B antibody, every ICeChIP was conducted with 12.5 uL of Protein A Dynabeads (Invitrogen) and 3 ug of antibody. The Protein A Dynabeads for each ICeChIP were washed with 50 uL of ChIP Buffer 1 by use of a magnetic rack, then resuspended in 50 uL of ChIP Buffer 1. In a separate set of tubes, 3 ug of each antibody was diluted to 100 uL with ChIP Buffer 1. The antibody suspension and the Protein A Dynabeads suspension were combined and incubated on a rotator at 4°C for at least 1 hour, then washed with 200 uL of ChIP Buffer 1 by use of a magnetic rack and resuspended in 50 uL of ChIP Buffer 1. After antibody washing, 150 uL of the diluted chromatin elution was added to each antibody-bead conjugate and incubated for 15 minutes on a rotator at 4°C. After this incubation, the beads were washed twice with 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, Filter Sterilized) and once with ChIP Buffer 3 (10 mM Tris pH 7.5, 250 mM LiCl, 1 mM EDTA, 0.5% Sodium Deoxycholate, 0.5% v/v NP-40 Substitute, Filter Sterilized), with a wash consisting of removal of the existing supernatant by use of a magnetic rack, resuspension into 150 uL of buffer, transfer to a new siliconized tube, and incubation on the rotator for 10 minutes at 4°C. After these washes, the supernatant was removed, and the beads were resuspended in ChIP Buffer 1, transferred to a new siliconized tube, then rinsed once with 200 uL of TE before being resuspended in 50 uL of ChIP Elution Buffer and incubated at 55°C for 5 minutes. After incubation, the supernatant was transferred to a new set of siliconized tubes, and the beads were discarded. To each supernatant was then added 2 uL of 5 M NaCl, 1 uL of 500 mM EDTA, and 1 uL of 10 mg/mL Proteinase K. 15 uL of Input DNA was also diluted to 50 uL with 35 uL of ChIP Elution Buffer (50 mM Tris pH 7.5, 1 mM EDTA, 1% w/v SDS, Filter Sterilized) and was supplemented with 2 uL of 5 M NaCl, 1 uL of 500 mM EDTA, and 1 uL of 10 mg/mL Proteinase K. The IP elutions and diluted input were then incubated at 55°C for 2 hours for a Proteinase K digestion. After digestion, the DNA was purified by adding 1.5 volumes of Serapure HD (1:50 dilution of Sera-Mag SpeedBeads [Fisher], 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 SpeedBeads), incubating at room temperature for 15 minutes, then collecting the beads on a magnetic rack, washing twice with 150 uL of 70% ethanol, and eluting into 50 uL of water, which was then recovered and stored at -20°C. DNA purified with Serapure was quantified using Quant-iT TM PicoGreen (Thermo Fisher) per manufacturer instructions. Up to 10 ng of each DNA sample for which libraries were to be prepared were then carried forward. Libraries were then generated from the NEBNext Ultra II DNA Library Prep kit (New England Biolabs) per manufacturer instructions. The DNA content of the library was then quantified and pooled for Illumina sequencing.