GSM3356426: 002_BAP-fused_macroH2A1_rep_2; Homo sapiens; MNase-Seq
Extraction of total (nonbiotinylated) and biotinylated chromatin from Hela S3 stably coexpressing BirA-emerin and BAP-macroH2A1 fusion proteins. HeLa S3 and HeLa BirA-emerin and BAP-macroH2A1 expressing cells were pulse labeled with 5 ug/ml biotin for 1 hour with the addition of 50 mM final HEPES added to stabilize pH. After the removal of the biotin, cells were washed with PBS following the washing in HB buffer (10 mM TrisHCl, pH 7.3, 10 mM KCl, 1.5 mM MgCl2 , 0.2 mM PMSF, 10 mM beta mercaptoethanol). Next, cells were incubated in HB buffer on ice for 20 min and nuclei were released by breaking the cells with size B pestle in dounce homogenizer and spun at 800g for 15 min. Nuclei were resuspended in an equal amount of the TGME buffer (50% Glycerol, 0.5 M Tris pH 7.9, 1M MgCl2, 500 mM EDTA, pH 8.0 and 5 mM DTT). For micrococcal digestion, the nuclei were resuspended in 500 uL of TM2 buffer (10 mM Tris-HCl, 2 mM MgCl2, 0.1% Triton, 1 mM PMSF), 2.5 uL of 0.5 M CaCl2 and 25 uL of micrococcal nuclease (MNase) (0.2 U/uL; Sigma; N3755) were added before incubation at 37°C for 10 min. The reaction was stopped by adding 15 uL of 0.1 M EGTA. Nuclei were collected by spinning at 400g for 10 min at 4°C. After MNase digestion, the nuclei pellet was resuspended in 500 uL of prechilled 0.4 M salt extraction buffer (385 mM NaCl, 10 mM Tris-HCl [pH 7.4], 2 mM MgCl2, 2 mM EGTA, 0.1% Triton X-100, 1 mM PMSF). The tubes were rotated at 4°C for 30 min. Supernatant containing digested chromatin was separated from the remaining material by spinning at 400g for 10 min at 4°C. An equal volume of TM2 buffer was added to the extracted chromatin to arrive at a 0.2 M final salt concentration. The sample was centrifuged at maximum speed for 5 min at 4°C. For the preclearing, three hundred μL of a suspension of Protein A agarose beads (Pierce 20334, Thermo Fisher) were washed three times with 0.2 M salt extraction. The bead suspension was combined with chromatin and rotated for 30 min at 4°C. The sample was centrifuged at maximum speed and supernatant transferred to a new tube. 300 μL of a suspension of sepharose-streptavidin beads (GE Healthcare; 17-5113-01) were washed three times with 0.2 M salt extraction buffer. The bead suspension was combined with the supernatant containing chromatin and rotated for 3 h at 4°C. Afterward, the beads were washed twice in 500 μL of 0.4 M salt extraction buffer containing Triton for 5 min at 4°C. DNA fraction was eluted twice by adding 100 µL of 1.5 M Ammonium bicarbonate and SpeedVac'ed. Afterwards, DNA was resuspended in 200 µL TE and an equal volume of phenol was added to sample and centrifuged for 5 min at maximum speed. Upper phase with DNA was transferred to a new tube. For library construction, DNA fragments (100 ng) were converted to blunt ends by end repair using Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific) and ligated to Ion Xpress Barcode Adapters.