organism: Homo sapiens
description: Protocols: 1,000,000 cells/sample were harvested from culture flasks by incubation with Trypsin-EDTA (Cat. # 25200056, Gibco) for 5 minutes. The trypsin was quenched with culture media and the cells were washed twice with DPBS (Cat. #14190094, Thermo Fisher Scientific). HCT116 human colorectal cancer cells were cultured in a humidified 37 °C incubator in 5% CO2. Culture medium consisted of high glucose Dulbecco's Modified Eagle Medium (Cat. #41965039, Gibco) with 10 % bovine calf serum (Cat. #1233C, Sigma-Aldrich) and 1X Penicillin-Streptomycin (Cat. #15140148, Gibco).  Nuclear extraction was performed by three washes Nuclear Extraction (NE) buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermidine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], Roche Complete Protease Inhibitor EDTA-Free). The nuclei were resuspended after the final wash in 1 ml wash buffer per sample and bound to 40 µl Magnetic ConA Agarose beads () equilibrated in binding buffer (HEPES pH 7.5 [20 mM], KCl [10 mM], CaCl2 [1 mM], MnCl2 [1 mM]). Bead binding proceeded for 15 min, after which nuclei and beads were resuspended in 200 µl wash buffer per sample and split into PCR tubes. Beads were collected on the magnet and then resuspended in 200 µl EDTA wash buffer (wash buffer with EDTA 2mM) and incubated RT for 5 min. Samples were then directly resuspended in antibody buffer (wash buffer with 2 ug antibody, 2 mM EDTA, 0.025% digitonin, and 0.05% BSA) and incubated ON at 4 °C on a rotator. After ON incubation, wash buffer always contained 0.025% digitonin. After ON incubation samples were washed 5 times and resuspended in 200 µl of Lazarus buffer (wash buffer with 480 ng Lazarus/sample) and incubated for 45 min at 4 °C on a rotator. Next, samples were washed five times and then resuspended in 100 ul biotinylation buffer (wash buffer with 5 mM MgCl2, 5 mM biotin, and 2 mM ATP) and incubated at 37 degrees for 30 min. Samples were washed twice to wash out excess biotin, chilled in ice for 5 min during the last wash, and then resuspended in 100 ul ice cold digestion buffer (wash buffer with 2 mM CaCl2). Digestion was allowed to proceed for 30 min at 4 degrees. Digestion was stopped by the addition of STOP mix (1 ul EDTA 500 mM, 1 ul EGTA 500 mM, 4 ul NaCl 5M). Samples were incubated at 37 degrees for 30 minutes, and then microfuged for 30 sec. The first release was transferred to new PCR tubes, and the beads resuspended in 50 ul 1X Urea STOP buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.8 M]) and incubated for 30 min at 20 degrees. After incubation, samples were microfuged for 30 sec and then the supernatant combined with the first release and the beads discarded. 20 ul Pierce streptavidin beads were prepared per sample by washing 3 times in PBS, and then resuspending in 50 ul PBS with 0.1 mM PMSF. Beads were added to each sample (final volume 200 ul) and inbubated at RT for 1 hr on a rotator. The beads were collected on a magnet, and supernatant containing DNA and non-biotinylated proteins was processed for DNA extraction (see DNA sequencing) and the beads attached to biotinylated proteins processed for mass spectrometry.  Supernatant from Pierce beads was transferred to new tubes containing 2 ul 10% SDS and 2.5 ul proteinase K, and incubated at 50 degrees for 1 hour. 1 volume of phenol:chloroform:isoamyl alcohol was added to each sample and vortexed for 30 sec. Samples were centrifuged at 15,000 x g for 15 min, and then the aqueous phase transferred to new tubes containing 1 ul glycogen, 20 ul 3M sodium acetate, and 500 ul 100% EtOH. Samples were incubated ON at -20 degrees, centrifuged at 15,000 x g at 4 degrees for 25, min, and the supernatant discarded. Pellets were washed once with 1 ml 70% EtOH, centrifuged again for 10 min, and then dried for 10 min at 50 degrees. Pellets were resuspended in 15 ul 10 mM Tris-HCl pH 8.0. Library preparation was performed with the KAPA EvoPrep kit and KAPA dual-index adapters according to manufacturer's instructions with the following modifications: 0.5X volumes were used throughout the protocol, libraries were amplified for 13 PCR cycles, and samples were size-selected with by cutting from 150 – 500 bp after running on 2% agarose E-gels with the Thermo Fisher E Gel machine.