High-Sensitivity ChIP Kit (ab185913)

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	Chromatin immunoprecipitation of FLAG-tagged MLL-AF9 was performed using the High Sensitivity ChIP Kit (Abcam, 185913) according to the manufacturer’s instructions.

Protocol tips
Chromatin immunoprecipitation of FLAG-tagged MLL-AF9 was performed using the High Sensitivity ChIP Kit (Abcam, 185913) according to the manufacturer’s instructions.

Publication protocol

MLL-AF9/NrasG12D AML cells and Drosophila melanogaster S2 cells were separately cross-linked with 10% formaldehyde and quenched with glycine (2.5 M). Pellets were washed, pooled, and resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0). Chromatin was sonicated to obtain fragments of 150 bp using a Bioruptor sonicator (Diagenode). 0.5% Triton X-100 was added to the samples to allow solubilization of the sheared DNA. Chromatin was incubated with antibodies overnight (5 µg each). Antibody-bound material was enriched using protein-G-coupled magnetic beads (Invitrogen), washed (50 mM Hepes-KOH, pH 7.4; 500 mM LiCl; 1 mM EDTA; 1% NP40 and 0,5% Na-Deoxycholate), and released using elution buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA and 1% SDS) at 65 °C. DNA-protein crosslinks were reverted by incubating the samples overnight at 65 °C in the presence of 0.2 M NaCl. The DNA was treated with RNaseA (0.2 mg/ml) and proteinase K (0.2 mg/ml) and purified using PCR clean-up kit (Qiagen). Chromatin immunoprecipitation of FLAG-tagged MLL-AF9 was performed using the High Sensitivity ChIP Kit (Abcam, 185913) according to the manufacturer’s instructions. Antibodies used were: anti-H3K4me3 (Abcam, 8580) anti-H3K36me3 (Abcam, 9050), anti-H3K79me2 (Abcam, 3594), anti-Flag (Sigma, F1804). Sequencing libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs) and sequenced on Illumina HiSeq 4000 using 50 bp single-read chemistry.

Raw ChIP-seq reads were evaluated with FastQC (version 0.11.4). Quality-filtering and trimming was done with PRINSEQ-lite (version 0.20.4). Resulting high-quality reads were simultaneously mapped against the Mus musculus (GRCm38) and Drosophila melanogaster (dm6) reference genomes via BWA (version 0.7.15). SAMtools (version 1.4) was used to split the alignments into mouse and Drosophila reads. Read normalization via the Drosophila melanogaster spike-in material was carried out with Deeptools (version 2.5.0.1) for each sample. Profile plots of histone marks were also generated with Deeptools (version 2.5.0.1). For the comparison of H3K79me2 vs. H3K36me3 signal intensities on MLL-target genes vs. non-MLL-targets, an equally sized set of randomly selected non-MLL-target genes was chosen. MLL-target genes represent genes that were downregulated upon MLL-AF9 withdrawal as measured by microarray analysis22. IGV was used for manual inspection and visualization of data. For the analysis of histone mark intensities in genes, mapped reads per gene were counted with featureCounts (1.5.0), respective input counts subtracted, and normalized via TMM using the edgeR package. The Pearson correlation coefficient between changes in respective histone marks over gene bodies after Setd2 knockdown was calculated with the functions bigwigCompare, multiBigwigSummary, and plotCorrelation of Deeptools.

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Manufacturer protocol

Download the product protocol from Abcam for High-Sensitivity ChIP Kit (ab185913) below.

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