harbouring a dox inducible CBFB-MYH11 knockdown construct
600 ng/ml doxycycline
CBFB sc-20693x (Santa Cruz)
Sequenced DNA Library
ChIP-seq: Purified cells were first cross-linked with 1% formaldehyde for 20 min at room temperature, quenched with 0.125 M glycine and washed with three buffers: (i) PBS, (ii) buffer of composition 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6 and (iii) 0.15 M NaCl, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6. Cells were then suspended in ChIP incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6) and sonicated using a Diagenode Bioruptor sonicator for 20 min at high power (30 seconds ON and 30 seconds OFF cycle). Sheared chromatin was centrifuged at maximum speed for 10 min and then incubated overnight at 4℃ in incubation buffer supplemented with 0.1% BSA with protein A/G-Sepharose beads (Santa Cruz) and 1 µg of antibody. Beads were washed sequentially with four different wash buffers at 4℃: two times with a solution of composition 0.1% SDS, 0.1% DOC, 1% Triton, 150 mM NaCl, TEE (10 mM Tris pH 8.0, 0.1 mM EDTA and 0.5 mM EGTA), one time with a similar buffer but now with 500 mM NaCl, one time with a solution of composition 0.25 M LiCl, 0.5% DOC, 0.5% NP-40, TEE and two times with TEE. Precipitated chromatin was eluted from the beads with 400 µl of elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature for 20 minutes. Protein-DNA crosslinks were reversed at 65℃ for 4 hours in the presence of 200 mM NaCl, after which DNA was isolated by Qiagen column. RNA-seq: Total RNA was isolated with the RNeasy RNA extraction kit with on-column DNaseI treatment (Qiagen), and the concentration was measured with a Qubit fluorometer (Invitrogen). Ribosomal RNA was removed by Ribo-Zero rRNA Removal Kit (Epicentre) according to manufacturer instructions. ChIP-seq: ChIP-seq libraries were prepared from precipitated DNA of 5 million cells (5-8 pooled biological replicas) for MLL, or from 1 million cells for the histone tail modifications. End repair was performed using Klenow and T4 PNK. A 3' protruding A base was generated using Taq polymerase and adaptors were ligated. The DNA was loaded on E-gel and a band corresponding to ~300 bp (ChIP fragment + adaptors) was excised. The DNA was isolated, amplified by PCR and used for cluster generation and sequencing on the HiSeq 2000 or NextSeq (Illumina). RNA-seq: Total RNA was isolated with the RNeasy RNA extraction kit with on-column DNaseI treatment (Qiagen), and the concentration was measured with a Qubit fluorometer (Invitrogen). Ribosomal RNA was removed by Ribo-Zero rRNA Removal Kit (Epicentre) according to manufacturer instructions. 16 µl of purified RNA was fragmented by addition of 4 µl 5× fragmentation buffer (200 mM Tris-acetate pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate) and incubated at 94℃ for exactly 90 s. After ethanol precipitation, fragmented RNA was mixed with 5 µg random hexamers, followed by incubation at 70℃ for 10 min and chilling on ice. We synthesized the first-strand cDNA with this RNA primer mix by adding 4 µl 5× first-strand buffer, 2 µl 100 mM DTT, 1 µl 10 mM dNTPs, 132 ng of actinomycin D, 200 U SuperScript III, followed by 2 h incubation at 48℃. First strand cDNA was purified by Qiagen mini elute column to remove dNTPs and eluted in 34 µl elution buffer. Second-strand cDNA was synthesized by adding 91.8 µl, 5 µg random hexamers, 4 µl of 5× first-strand buffer, 2 µl of 100 mM DTT, 4 µl of 10 mM dNTPs with dTTP replaced by dUTP, 30 µl of 5× second-strand buffer, 40 U of Escherichia coli DNA polymer¬ase, 10 U of E. coli DNA ligase and 2 U of E. coli RNase H, and incubated at 16℃ for 2 h followed by incubation with 10 U T4 polymerase at 16℃ for 10 minutes. Double-stranded cDNA was purified by Qiagen mini elute column and used for library preparation as described in the KAPA HyperPrep protocol.