CHRF
Homo sapiens
Protocols: CHRF 288-11 cells were cultured in RPMI-1640 (Sigma-Aldrich, Gillingham, UK, R0883), 10 % FBS (Invitrogen, Paisley, UK, 10100-147) and 200 uM L-glutamine, 100 U penicillin and 100 ug/ml streptomycin (Sigma-Aldrich, G1146). 20 million CHRF cells per condition were crosslinked for 10 minutes with 1% formaldehyde. Cells were lysed for 10 minutes on ice (10 mM Tris pH8.0, 10 mM NaCl, 0.2% NP-40, Complete). Nuclei were lysed with for 10 minutes on ice (50 mM Tris pH8.1, 10 mM EDTA, 1% SDS, Complete). Crosslinked chromatin was sheared for 2x5 minutes by sonication using a Bioruptor (Diagenode, Cambridge, UK) and pre-cleared with 100 ug anti-rabbit IgG pre-immune serum (Sigma-Aldrich I5006) using protein G sepharose beads (Roche Diagnostics, Burgess Hill, UK, 11 243 233 001). Chromatin immunoprecipitation was performed over night in IP dilution buffer (20 mM Tris pH8.1, 2mM EDTA, 150 mM NaCl, 1% Triton X100, 0.01% SDS), using 14 ug anti-Meis1 (Abcam, Cambridge, UK, ab19867, Lot No. 153899). Beads were washed twice with wash buffer 1 (20 mM Tris pH 8.1, 2 mM EDTA, 50 mM NaCl, 1 % Triton X100, 0.1 % SDS), once with wash buffer 2 (10 mM Tris pH 8.1, 1 mM EDTA, 250 mM LiCl, 1% NP40, 1% sodium deoxycholate monohydrate) and twice with TE buffer. Bound chromatin was eluted from the beads twice with 100 mM NaHCO3, 1 % SDS. After reverse-crosslinking, RNaseA and proteinase K digestion, chromatin was cleaned up using the Qiagen PCR purification kit (Qiagen, Crawley, UK, 28104).