Yeast strains were grown to saturation in YPD before being diluted to an OD600 of 0.2 and grown to an OD600 of ~1 at 30°C. For nutrient deprivation experiments, cells were isolated, washed with water, and resuspended in SD media (2% glucose, 0.15% yeast nitrogen base, 0.5% ammonium sulfate) (Cheung et al., 2008 (link)). Fifty ODs of cells were collected at each time point and ChIP was performed as described with modifications (Ahn et al., 2009 (link)). For precipitation of proteins, 2 ul of H3K36me1 (abcam 9048), H2K36me2 (Active Motif 39255), H3K36me3 (abcam 9050), H3K27ac (Active Motif, 39133), H3K56ac (Active Motif, 39281), or H3 C-terminal (EpiCypher, 13–0001) was used and IgG (Cell Signaling 2729) was the negative control. DNA was eluted in 30 μL of elution buffer (Zymo) and diluted 1:20. Two μl of the diluted DNA was subjected to qPCR using SYBR Green (Bio-Rad) and primers described in Table S3 .
H3k36me1
Manufactured by Abcam
Sourced in United States, United Kingdom
H3K36me1 is a histone H3 methylation mark that is associated with active transcription. It is a post-translational modification of histone H3 at lysine 36 where a single methyl group is added. This modification plays a role in regulating gene expression patterns.
Automatically generated - may contain errors
Lab products found in correlation
H3k36me3, by Abcam (5 mentions)
H3k36me2, by Active Motif (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
H3k36me2, by Cell Signaling Technology (2 mentions)
Histone h3, by Abcam (2 mentions)
H3k56ac, by Active Motif (1 mentions)
H3k27ac, by Active Motif (1 mentions)
Elution buffer, by Zymo Research (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Ab37910, by Abcam (1 mentions)
Ab4729, by Active Motif (1 mentions)
Ab75359, by Abcam (1 mentions)
Ab9050, by Abcam (1 mentions)
Ab6002, by Abcam (1 mentions)
H3k27me3, by Abcam (1 mentions)
Cleaved parp 1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 7, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
H3k27ac, by Abcam (1 mentions)
H3k36me2, by Merck Group (1 mentions)
8 protocols using h3k36me1
1
Chromatin Immunoprecipitation for Histone Modifications
2
Apoptosis, Growth, and Gene Expression Assays
3
Whole-Cell Yeast Extract Preparation
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Whole-cell extracts for yeast were prepared according to protocol published elsewhere25 (link). Briefly, 5 ml yeast cultures grown to the mid-log phase were collected, washed once with water and resuspended in 400 μl of 2 M NaOH with 8% β-mercaptoethanol. The suspension was incubated on ice for 5 min, followed by a 10 min spin at 16,000 g. The supernatant was removed and the pellet was resuspended in 400 μl of Buffer A (40 mM HEPES-KOH pH 7.5, 350 mM NaCl. 0.1% Tween-20, 10% glycerol and protease inhibitors) incubated in ice for 10 min and harvested as above. The resultant pellet was resuspended in 2 × SDS loading dye and separated on a 15% SDS–PAGE gel. The gel was transferred to an activated PVDF membrane using the wet-transfer method. Antibodies used for chromatin immunoprecipitation (ChIP) and western blotting are H3 (Abcam, #1791), H3K36me3 (Abcam, #9050) H3K36me2 (Millipore, #07–369) and H3K36me1 (Abcam, #9048). All the antibodies were used at a dilution of 1:1,000 for the western blot. Original uncropped blots of all western blots used for the figures are shown in Supplementary Fig. 9 .
4
Western Blot Analysis of Histone Methylation
5
Immunoblotting for EMT and Stemness
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Primary antibodies used are CDH1 (Cell Signaling Technology, Beverly, MA, USA; #3195S), MUC1 (Abcam, Boston, MA, USA; #ab109185), SNAI2 (Cell Signaling Technology; #9585S), MMP2 (Proteintech, Rosemont, IL, USA; #10373‐2‐AP), CD44 (GeneTex, Irvine, CA, USA; GTX102111), CD34 (Proteintech; 60180), vimentin (Santa Cruz Biotechnology; B0719), total SMAD2 (Cell Signaling Technology; #3103S), phosphorylated SMAD2 (Cell Signaling Technology; #3108S), TGF‐β (Cell Signaling Technology; #3711S), H3K36me1 (Abcam; #ab9048), H3K36me2 (Cell Signaling Technology; #2901S), H3K36me3 (Active Motif; #61101), histone H3 (Abcam; #ab1791), SOX2 (R&D Systems; #AF2018‐SP), OCT2 (Thermo Fisher; #39‐5400), PRRX1 (Novus Biologicals, St. Charles, MO, USA; #NBP1‐06067), anti‐FLAG (Sigma‐Aldrich; #F1804), GAPDH (Cell Signaling Technology; #2118S), and lamin B1 (Proteintech; #12987‐1‐AP). Secondary antibodies are anti‐rabbit (Invitrogen, Waltham, MA, USA; #SA5‐3557) and anti‐mouse (LI‐COR, Lincoln, NE, USA; #926‐68070). Lentiviral vectors are eGFP (control vector; Genecopoeia; EX‐EGFP‐Lv181), SOX2 (Genecopoeia; EX‐T2547‐Lv181), OCT2 (Genecopoeia; EX‐A2204‐Lv181), and PRRX1 (Genecopoeia; EX‐T1345‐Lv181).
6
Chromatin Immunoprecipitation Protocols with Histone Modifications
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The antibodies used for ChIP-qPCR were anti-H3K9me1 (EpiCypher, 13-0014; Abcam, 8896), H3K9me3 (Abcam, 8898; Active Motif, 39765), H3K56me1 (Abcam, 66857), H3K56me2 (Active Motif, 39277), H3K36me3 (Abcam, 9050; Cell Signaling Technology, 4909S), H3K36me1 (Abcam, 176920), Histone H3 (Abcam, ab1791), and γH2Av (mouse; Hybridoma Bank, UNC93-5.2.1). The primary antibodies used for IF were anti-γH2Av (mouse; Hybridoma Bank, UNC93-5.2.1), anti-Rad51 (a kind gift from J. Kadonaga), and anti-Cyclin A (mouse; 1:10; Developmental Studies Hybridoma Bank, A12). The secondary antibodies used for IF were Alexa 488/568/647 goat anti-mouse (1:1000; Thermo Fisher Scientific).
7
Immunoprecipitation and Western Blot Analysis of Histone Methylation
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HEK293 cells were obtained from German Collection of Microorganisms and Cell Cultures GmbH (https://www.dsmz.de/). They were grown in Dulbecco's Modified Eagle's Medium (Sigma) supplemented with 5% fetal bovine serum, penicillin/streptomycin, and L-glutamine (Sigma) in an incubator providing 37 °C and 5% CO 2 . The pECFP-C1 tagged full-length NSD2 was co-transfected with pEYFP-C1 fused ATRX or FANCM using polyethylenimine (Polyscience, USA; according to manufacturer´s instructions). 72 h after transfection, the cells were washed with PBS buffer and harvested by centrifugation at 500 g for 5 min. For methylation analysis, the YFP-fused ATRX and FANCM substrate proteins were immunoprecipitated from cell extract using GFP-Trap® A (Chromotek) following the manufacturer´s instructions. The samples were heated to 95 °C for 5 min in SDS-gel loading buffer and resolved by 16% SDS-PAGE. Analysis was performed by Western Blot using as primary antibody H3K36me1 (Abcam, UK; Cat. No: ab9048) or GFP antibody (Clontech, lot. 1404005).
8
PRDM9-mediated histone modifications
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A FLAG-tagged control vector or FLAG-tagged PRDM9 construct were transfected into HEK293 cells using GeneJuice (Novagen). Transfected cells were serum-deprived prior to lysis in 50 mm Tris-HCl, 400 mm NaCl, 1 mm EDTA, 0.1% SDS, and 1% Triton X-100 supplemented with protease inhibitors. 30 μg of total cell lysate were processed for Western blotting and immunoblotted using specific antibodies to monitor the levels of H3K36me1 (Abcam, ab9048), H3K36me2 (Cell signaling Technology, 2901), H3K36me3 (Cell Signaling Technology, number 9763), H3K4me1 (Cell Signaling Technology, 9723), H3K4me2 (Abcam, ab32356), H3K4me3 (Cell Signaling Technology, number 9727S), and total H3 (Abcam, ab10799). The transfection efficiency of FLAG-PRDM9 was controlled by using an anti-FLAG antibody (FLAG M2 monoclonal, Sigma). Membrane bands were visualized using LiCor IRDye, 680RD anti-rabbit and 800CW anti-mouse secondary antibodies.
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