ChIP-Seq of Homo Sapiens: mantle cell lymphoma : Sample MCL-02
Frozen blocks of tumor tissue were trimmed as needed to remove low tumor cellularity regions. Frozen sections (25 micron) were then cut to a total of ~50 mg of tissue for each ChIP-Seq chromatin prep. For crosslinking, sections were dissociated in PBS + PI + 10 mM Na butyrate, formaldehyde was added to 1% and the sample incubated for 10 minutes at 37°C, quenched for 5 minutes with 125 mM glycine, washed twice in cold PBS with protease inhibitors, and stored at -80°C.  Samples were lysed in a 200-500 mcl of ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1), diluted to 900 mcl in ChIP dilution buffer 1 (0.01% SDS, 0.25% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl), and sonicated to an average fragment size of 200-400 bp on a Branson sonifier, followed by dilution with ChIP dilution buffer 2 (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl) to a final dilution of 1:10 with respect to the original lysis volume. DNA was quantified from sonicated chromatin, and an equivalent of 2-5 million cells were used per ChIP, with 25-50 mcl of input chromatin saved as a control. Cells were rotated at 4°C overnight with 2-5 ug of H3K27ac antibody (39133, Active Motif). Antigen-antibody complexes were collected with protein G Dynabeads (Life technologies) for 4 hours at 4 C, and sequentially washed with RIPA buffer (0.1% Na Deoxycholate, 0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 140 Mm NaCl), RIPA / High salt buffer (0.1% Na Deoxycholate, 0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 360 Mm NaCl), LiCl Wash Buffer (250mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.0), and TE Buffer pH 8.0 (10mM Tris-HCl pH 8.0, 1mM EDTA). Washed beads or unenriched input chromatin were resuspended in Low-SDS ChIP elution buffer (10mM Tris-HCl pH 8.0, 0.5M EDTA, 300mM NaCl, 0.1% SDS, 5mM DTT) and incubated for 6 hours at 65°C to elute DNA and reverse crosslinking. Supernatants were treated with RNase and proteinase K, and ChIP DNA was then purified with AMPure beads (Beckman-Coulter). For sequencing library preparation, ChIP DNA was end-repaired (End-It, Epicentre), A-tailed (Klenow fragment 3'-->5' exo-, New England Biolabs), and ligated to barcoded illumina adaptors (Quick T4 DNA ligase, NEB). Each reaction was followed by clean-up with AMPure beads. Ligation products were amplified by PCR for 14-18 cycles with Illumina primers and PFU Ultra II HS PCR mix (Agilent). Library size selection to 300-600 bp was performed by gel purification (E-Gel SizeSelect 2%, Life technologies) or two-step AMPure bead selection.  Paired-end sequencing of H3K27ac ChIP and an unenriched chromatin control library was performed on a HiSeq 2500 or NextSeq 500. Reads were aligned to hg19 using BWA-ALN.
MCL-02 H3K27ac