Ab9050

For chromatin immunoprecipitation, HepG2 cells were cross-linked with 1% formaldehyde at 37°C for 10 min and quenched with 0.125 M glycine for 5 min at room temperature. ChIP was performed using a ChIP assay kit (Millipore) according to the manufacturer’s protocol. Antibodies used for ChIP were GFP (gta-20, Chromo-Tek) and H3K36me3 (ab9050, abcam).

ChIP was performed as described [56 (link),57 (link)]. Briefly, worm pellets were ground with a mortar and pestle and cross-linked with 1% formaldehyde in PBS for 10 mins at room temperature. Worm fragments were collected by spinning at 3,000g for 5 mins and resuspended in FA buffer followed by sonication with Bioruptor to generate chromatin fragments with major DNA length around 200 bp. Chromatin extracts were incubated with H3 (rabbit; Abcam, ab1791) or H3K4me3 antibodies (rabbit; Abcam ab9050) overnight at 4°C. Antibodies used were prescreened for specificity using dot blots. The optimal amounts of antibodies used were determined by titration in a preliminary experiment with ChIP-qPCR. Precipitated DNA (10–15 ng) from each sample was used for Illumina sequencing library preparation. DNA from ChIP was first end-repaired to generate a blunt end followed by adding single adenine base for adaptor ligation. The ligation products with adaptors were size-selected and amplified by PCR with primers targeting the adaptor. Up to 12 samples were multiplexed in one lane for single-end 50-nt Illumina HiSeq sequencing or single-end 75-nt Illumina NextSeq500. Raw ChIP-seq data have been deposited at Gene Expression Omnibus (GSE101964).

Immunohistochemical analysis was performed with 4-μm-thick FFPE tumor tissue sections. Briefly, after deparaffinization, antigen retrieval was performed for 30 min in citrate buffer (pH 6.0). The slides were then incubated with the following primary antibodies: H3 K27M (Millipore, ABE419, 1:500), H3K36 trimethylation (Abcam, ab9050, 1:2000).

Gametocytes were treated with JIB-04 (5 µM) on days 2, 3 and 4 of development and sampled 24 h later. Histones were extracted as described before [17 (link)] with minor modifications. Nuclei were extracted by Dounce homogenisation in hypotonic lysis buffer (10 mM Tris–HCl (pH 8), 0.25 M sucrose, 3 mM MgCl₂, 0.2% (v/v) Nonidet P-40) and protease inhibitors (Roche)). Histones were isolated from nuclei resuspended in Tris buffer (10 mM Tris (pH 8.0), 0.8 M NaCl, 1 mM EDTA and protease inhibitors) by overnight acid-extraction (0.25 M HCl, with rotation at 4 °C) and subsequent precipitated with 20% TCA. Histone pellets were washed with acetone, reconstituted in dddH₂O and spotted quantitatively (100 ng per sample) on nitrocellulose membranes. Membranes were submerged in blocking buffer for 30 min followed by 1 h incubation with α-H3K36me2 (Abcam, ab9049) or α-H3K36me3 (Abcam, ab9050) primary antibody dilutions (1:5000 in TBS-t). Membranes were washed three times in TBS-t and then incubated with goat α-rabbit IgG antibody conjugated to HRP (1:10000) for 1 h. Chemiluminescent signal (Pierce SuperSignal West Pico PLUS Chemiluminescent Substrate) was quantified with ImageJ analysis software [95 (link)].

Cells were fixed in 100% ice-cold methanol at −20 °C for 10 min, washed three times with 1X PBS and further permeabilized in 0.3% Triton X-100 for 10 min. Following blocking in 10% normal goat serum (MP Biomedicals), cells were incubated with the primary antibody in blocking buffer at 4 °C overnight. The following antibodies were used for immunofluorescence: H4/H2AS1ph (ab177309, Abcam; 1:2000), Lamin A/C (ab238303, Abcam; 1:1000), H3K4me3 (ab8580, Abcam; 1 μg/ml) and H3K36me3 (ab9050, Abcam; 1 μg/ml). Next, cells were washed three times with 1X PBS and following incubation with Alexa Fluor 568 goat anti-rabbit (A11011, Thermo Fisher Scientific; 1:1000) and Alexa Fluor 488 goat anti-mouse (A11001, Thermo Fisher Scientific; 1:1000) secondary antibodies diluted in 10% normal goat serum for 1 h at room temperature, nuclei were stained with DAPI (Dako) or Hoechst 33342 (Invitrogen). Samples were imaged on a ZeissAxio Observer.A1 microscope. For confocal and super resolution microscopy imaging was carried out on a ZEISS LSM 900 with Airyscan 2 using Zen blue for acquisition and processing. Airyscan2 images were processed using the default deconvolution settings and histogram stretching, applied when required, was identical between control and treated samples for each channel.