IgG-R
BioProject of this experiment was changed from PRJDB1761 to PRJDB1759.
BmN4 cells was cross-linked with 1% formaldehyde at room temperature for 10 min, and the cross-linking was stopped with a glycine stop-fix solution (PBS, glycine buffer) for 5 min at room temperature. The cells were washed twice with cold PBS and harvested. The cells were lysed in 5 mL of lysis buffer 1 (50 mM Hepes-KOH at pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100). The lysate was incubated at 4 degree_C for 10 min and centrifuged at 1500 rpm for 5 min at 4 degree_C. The pellet was then resuspended in 5 mL of lysis buffer 2 (10 mM Tris-HCl at pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA). The cell lysate was incubated at room temperature for 10 min and centrifuged at 1500 rpm for 5 min at 4 degree_C. The pellet was resuspended in 1 mL of lysis buffer 3 (10 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, and 0.5% N-lauroylsarcosine). Next, the lysate was sonicated on ice for 16 30-sec cycles using a sonicator (TOMY SEIKO). A 100-microL aliquot of 10% Triton-X 100 was added, and the cell lysate was centrifuged at 14,000 rpm for 10 min. A 50-microL portion of the supernatant was saved as whole-cell extract (WCE) DNA. The supernatant was then mixed with washed magnetic beads bound to 10 microg of RNA Polymerase CTD repeat monoclonal antibody (Abcam: ab817), monoclonal anti-H3K4me3 antibody (Abcam: ab1012), or polyclonal anti-H3Ac antibody (Millipore, 06-599). The samples were rotated at 4 degree_C overnight and washed eight times with 1-mL aliquots of wash buffer (50 mM Hepes-KOH at pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% Na-deoxycholate) and once with TE buffer containing 50 mM NaCl. Elution buffer (200 microL) was added, and the sample was eluted at 65 degree_C for 15 min. The supernatant was transferred to a new tube and incubated at 65 degree_C overnight. Elution buffer (150 microL) was added to the WCEmicroDNA, and the reaction was incubated at 65 degree_C overnight. Approximately 200 microL of TE was added to the IP and WCEmicroDNA samples. An 8-microL aliquot of 10 mg/mL RNase A (Funakoshi) was added, and the reactions were incubated at 37 degree_C for 2 h. A 4 microL-portion of 20 mg/mL proteinase K (Takara) was added, and the reactions were incubated at 55 degree_C for 2 h. The DNA was recovered by a phenol chloroform extraction and ethanol precipitation. Using the recovered DNA, the samples were prepared for the Illumina GA according to the manufacturer's instructions.