Illumina HiSeq 2500 sequencing; A genome-wide analysis of the distribution of H2A.XY39ph in human breast cancer cell line SUM159
A genome-wide analysis of the distribution of H2A.XY39ph in human breast cancer cell line SUM159
Cells are fixed in formaldehyde (or some other crosslinking reagent), such that any proteins that are bound to or near DNA are covalently linked to the chromatin.  The fixation reaction is then quenched with addition of glycine, and the cells are harvested and spun down in ice-cold PBS 1. Resuspend the cell pellet in 10 mL of cell lysis buffer for ChIP (cold) and incubate for 10 min on ice.  2. Pellet the cell nuclei at 1000 rpm in a benchtop centrifuge for 10 min at 4°C. Aspirate the supernatant carefully.  3.Resuspend the cell nuclei in 500 μL of MNase digestion buffer. Add MNase and incubate for 1 h on ice. Stop the digest by adding EDTA to a final concentration of 50 mM.  The amount of MNase will need to be optimized according to final chromatin size in Step 8. A range of 50-300 U is a convenient starting point. 4. Resuspend the nuclear pellet in 1 mL of nuclei lysis buffer for ChIP supplemented with protease inhibitors and incubate for 10 min on ice.  5. Proceed with sonication. Ensure that the samples do not foam and are kept as cold as possible.  Sonication conditions will need to be optimized. Typically, six 15-sec pulses followed by 45-sec rest periods at output 6.0 have been found to work. It is helpful to analyze each sample for proper sonication. For this analysis, reverse the cross-links and purify a small volume of chromatin as described below, and then analyze on an agarose gel. DNA should ideally be between 300 and 1000 bp in size. 6. Transfer chromatin to a 1.5-mL microcentrifuge tube and freeze at −80°C for storage. 7. Thaw the chromatin on ice. Centrifuge samples at high speed in a microcentrifuge for 15 min at 4°C to pellet the precipitated SDS. Transfer the supernatant to a fresh microcentrifuge tube.  At this step, it is helpful to aliquot chromatin for storage at −80°C. 8. Measure the DNA concentration (A260) of the chromatin using a spectrophotometer. Use nuclei lysis buffer for ChIP devoid of SDS as a blank.  Typically, the chromatin concentration should be >750 ng/μL. The A260/A280 ratio should be ~1.4-1.6. 9. Aliquot 100 μg of chromatin per antibody to be used into microcentrifuge tubes.  It is important to have an irrelevant antibody such as α-glutathione S-transferase as a control. 10. Dilute the chromatin to a final volume of 300 μL with dilution buffer supplemented with protease inhibitors.  11. To preclear the chromatin, add 50 μL of protein A-agarose/salmon sperm DNA or protein G-agarose/salmon sperm DNA beads to the chromatin and rotate for 1-2 h at 4°C.  Use large-orifice tips or cut off the end of regular micropipettor tips for transfer of beads. 12. Centrifuge the chromatin samples at 3000 rpm for 5 min at 4°C. Transfer the supernatants to fresh microcentrifuge tubes.  13. Add relevant antibodies to the chromatin samples and rotate overnight at 4°C. 14. Add 50 μL of protein A-agarose/salmon sperm DNA or protein G-agarose/salmon sperm DNA (depending on antibody) beads to the chromatin samples on ice. Rotate for 2 h at 4°C.  Make sure bead slurry is completely resuspended before adding to samples. 15. Centrifuge the samples at 3000 rpm for 2 min at 4°C.  16. Collect 15 μL from the supernatant of the control antibody sample as a 5% input. Keep this sample on ice for later use.  17. Carefully aspirate the supernatants of all samples.  Be careful not to aspirate any beads. Narrow gel-loading tips are useful for careful aspiration. 18. Add 1 mL of high-salt wash buffer for ChIP to all samples and rotate for 10 min at room temperature.  19. Centrifuge the samples at 3000 rpm for 2 min at room temperature.  20. Carefully aspirate the supernatants and add 1 mL of high-salt wash buffer for ChIP. Rotate for 10 min at room temperature.  21. Repeat Steps 22-23 twice for a total of four high-salt washes.  22. Aspirate supernatants and wash twice with TE as above.  23. Resuspend beads and input samples in 300 μL of elution buffer for ChIP supplemented with 1 μL of proteinase K (20 μg/μL), and incubate samples for 2 h at 55°C.  Do not resuspend beads by pipetting. Instead, gently flick or vortex samples. 24. To reverse cross-links, incubate overnight at 65°C. For library preparation was performed by the BerryGenomics Company using Illumina HiSeq2500 V4 with 50-bp paired-end reads.