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H3k9me2 3

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H3K9me2/3 is a specific antibody that recognizes dimethylated and trimethylated lysine 9 on histone H3. This modification is associated with gene silencing and heterochromatin formation.

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Lab products found in correlation

Nebnext ultra 2 dna library prep kit, by New England Biolabs (3 mentions) D4w1u, by Cell Signaling Technology (3 mentions) Megascript sp6 kit, by Thermo Fisher Scientific (1 mentions) H3k4me2 3, by Abcam (1 mentions) Nanoject microinjector, by Drummond (1 mentions) P entry cloning system, by Thermo Fisher Scientific (1 mentions) H3k27me3, by Cell Signaling Technology (1 mentions) Histone 3, by Cell Signaling Technology (1 mentions) Triptolide, by Merck Group (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Sfrp1, by Cell Signaling Technology (1 mentions) Wnt3a, by Cell Signaling Technology (1 mentions) H3k27me1, by Cell Signaling Technology (1 mentions) P erk, by Cell Signaling Technology (1 mentions) Flag m2, by Merck Group (1 mentions) P akt, by Cell Signaling Technology (1 mentions) Fgfr4, by Cell Signaling Technology (1 mentions) H3k36me3, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Fibroblast growth factor 2 (fgf2), by Cell Signaling Technology (1 mentions)

Spelling variants of this product

H3K9me2 (42 citations) Anti-H3K9me2/3 (3 citations)

6 protocols using h3k9me2 3

1

Overexpression of Chromatin Modifiers

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Mouse KDM6B (aa1025–1642) or KDM5B (aa1–770) were cloned using the p-Entry cloning system (Invitrogen, #K2400-20 and 11791-020) into a pCS2+ plasmid with a C-terminal HA-tag and NLS-tag. mRNA was synthesized in vitro using a MEGAscript SP6 Kit (Ambion, AM1330M) following the manufacturer's instructions. Eggs were in vitro–fertilized and de-jellied using a 2% cysteine solution in 0.1 × MMR. Injections into one-cell stage embryos were performed in injection solution (Smith et al. 2006 (link)) using a Drummond Nanoject microinjector, delivering 9.2 ng of mRNA per injection (mRNA at 1 mg/mL in DEPC H2O). Embryos were cultured at 18°C and collected for Western blot analysis at stage 21 (Nieuwkoop and Faber 1994 ). Western blot analyses were performed on 12% polyacrylamide gels using antibodies against H3K27me3 (Cell Signalling, #9733), H3K9me2/3 (Cell Signalling, #5327), H3K4me2/3 (Abcam, #8580), H4 (Abcam, #31830), and against H3 (Abcam, #18521)
Teperek M., Simeone A., Gaggioli V., Miyamoto K., Allen G.E., Erkek S., Kwon T., Marcotte E.M., Zegerman P., Bradshaw C.R., Peters A.H., Gurdon J.B, & Jullien J. (2016). Sperm is epigenetically programmed to regulate gene transcription in embryos. Genome Research, 26(8), 1034-1046.
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2

Nuclei Immobilization and Chromatin Cleavage

Cited 1 time
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A detailed protocol can be found on protocol.io from the Henikoff lab (https://doi.org/10.17504/protocols.io.mgjc3un; Skene et al., 2018 (link)). Briefly, 2 million cell nuclei were immobilized on Concanavalin A beads after washing. SALL4 (CST D16H12) or H3K9me2/3 (Cell Signaling D4W1U) antibodies, or normal rabbit IgG (Cell Signaling DA1E) were incubated with the nuclei overnight in the presence of 0.02% digitonin at 4 degrees. The next day, 700ng/mL of proteinA-micrococcal nuclease (pA-Mnase purified in house with vector from Addgene 86973, protocol from Schmid et al., 2004 (link)) were incubated with the nuclei at 4 degrees for an hour. After washing, the tubes were placed in heat blocks on ice set to 0 degrees, CaCl2 (1mM) was added and incubated for 30 min before 2× Stop buffer containing EDTA was added. DNA was eluted by heat and high-speed spin, then phenol-chloroform extracted. Qubit was used to quantify purified DNA and Bioanalyzer (2100) traces were run to determine the size of the cleaved products. NEBNext Ultra II DNA library prep kit (NEB E7645) was used to make the libraries according to Liu et al.’s protocol, outlined on protocols.io (https://doi.org/10.17504/protocols.io.wvgfe3w; Liu et al., 2018b (link)). Pair-end (42bp) Illumina sequencing was performed on the bar-coded and amplified libraries.
Kong N.R., Bassal M.A., Tan H.K., Kurland J.V., Yong K.J., Young J.J., Yang Y., Li F., Lee J.D., Liu Y., Wu C.S., Stein A., Luo H.R., Silberstein L.E., Bulyk M.L., Tenen D.G, & Chai L. (2021). Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression. Cell reports, 34(1), 108574.
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3

Antibody-based Protein Expression Analysis

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Antibodies to β-catenin, WIF1, Histone 3, β-actin, Wnt3a, H3K4me2&3, H3K9me2&3, H3K27me1, 2&3, H3K36me2&3, H3K79 2&3, SFRP1, and DKK1 were purchased from Cell Signaling. Antibodies to FRZB and ENY2 were purchased from Abcam. Rabbit IgG, Mouse IgG were purchased from Abcam. Triptolide was purchased from Sigma.
Nardi I., Reno T., Yun X., Sztain T., Wang J., Dai H., Zheng L., Kim J, & Raz D. (2018). Triptolide inhibits Wnt signaling in NSCLC through upregulation of multiple Wnt inhibitory factors via epigenetic modifications to Histone H3. International journal of cancer, 143(10), 2470-2478.
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4

Nuclei Immobilization and Chromatin Cleavage

Cited 1 time
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A detailed protocol can be found on protocol.io from the Henikoff lab (https://doi.org/10.17504/protocols.io.mgjc3un; Skene et al., 2018 (link)). Briefly, 2 million cell nuclei were immobilized on Concanavalin A beads after washing. SALL4 (CST D16H12) or H3K9me2/3 (Cell Signaling D4W1U) antibodies, or normal rabbit IgG (Cell Signaling DA1E) were incubated with the nuclei overnight in the presence of 0.02% digitonin at 4 degrees. The next day, 700ng/mL of proteinA-micrococcal nuclease (pA-Mnase purified in house with vector from Addgene 86973, protocol from Schmid et al., 2004 (link)) were incubated with the nuclei at 4 degrees for an hour. After washing, the tubes were placed in heat blocks on ice set to 0 degrees, CaCl2 (1mM) was added and incubated for 30 min before 2× Stop buffer containing EDTA was added. DNA was eluted by heat and high-speed spin, then phenol-chloroform extracted. Qubit was used to quantify purified DNA and Bioanalyzer (2100) traces were run to determine the size of the cleaved products. NEBNext Ultra II DNA library prep kit (NEB E7645) was used to make the libraries according to Liu et al.’s protocol, outlined on protocols.io (https://doi.org/10.17504/protocols.io.wvgfe3w; Liu et al., 2018b (link)). Pair-end (42bp) Illumina sequencing was performed on the bar-coded and amplified libraries.
Kong N.R., Bassal M.A., Tan H.K., Kurland J.V., Yong K.J., Young J.J., Yang Y., Li F., Lee J.D., Liu Y., Wu C.S., Stein A., Luo H.R., Silberstein L.E., Bulyk M.L., Tenen D.G, & Chai L. (2021). Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression. Cell reports, 34(1), 108574.
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5

Comprehensive Antibody and Growth Factor Panel

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The source of antibodies used in the study were as follows: AbCam H3K9ac (ab4441), H3K27Ac (ab4729). Cell Signaling Technologies AKT (9272), p-AKT (4056, 4058), CTBP1 (8684), ERK (9102), p-ERK (9101), FGFR4 (8562), GAPDH (2118), H3K4me3 (9751), H3K4me2 (9725), H3K36me3 (4909), H3K9me2/3 (5327), HA-tag (3724), HDAC1 (5356), HDAC2 (57156), Histone H3 (14269), JUN (9165), LSD1 (2139), MEK (4694), p-MEK (9154), pan-RAS (3965), RBBP7 (9067), RCOR1 (14567), SIN3A (8056), Stretavidin-HRP (3999). All CST antibodies were used at 1:1000 in 5% milk/PBST buffer. Santa Cruz c-MYC (sc-4084), HRAS (sc-34), KRAS (sc-30), NRAS (sc-519), TUBULIN (sc-69969). Santa Cruz antibodies were used at 1:500 except for TUBULIN at 1:3000. Sigma Aldrich FLAG-M2 (F1804, 1:1000), FGFR4 (HPA-028251, 1:500), RREB1 (HPA-001756, 1:500). The source of growth factors and mitogens were as follows: Cell Signaling Technologies EGF (8916), FGF1 (5234), FGF2 (61972), TGF-beta (8915), TNF-alpha (8902). Origene FGF8 (TP723101). Prospec HGF (cyt-244), FGF16 (cyt-939). Mek2 was a gift from Dustin Maly (Addgene plasmid # 40776; http://n2t.net/addgene:40776; RRID:Addgene_40776).
Kent O.A., Saha M., Coyaud E., Burston H.E., Law N., Dadson K., Chen S., Laurent E.M., St-Germain J., Sun R.X., Matsumoto Y., Cowen J., Montgomery-Song A., Brown K.R., Ishak C., Rose J.L., De Carvalho D.D., He H.H., Raught B., Billia F., Kannu P, & Rottapel R. (2020). Haploinsufficiency of RREB1 causes a Noonan-like RASopathy via epigenetic reprogramming of RAS-MAPK pathway genes. Nature Communications, 11, 4673.
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6

Mapping Chromatin Accessibility and Histone Modifications

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A detailed protocol can be found on protocol.io from the Henikoff lab (dx.doi.org/10.17504/protocols.io.mgjc3un) (Skene et al., 2018) . Briefly, 2 million cell nuclei were immobilized on Concanavalin A beads after washing. SALL4 (CST D16H12) or H3K9me2/3 (Cell Signaling D4W1U) antibodies, or normal rabbit IgG (Cell Signaling DA1E) were incubated with the nuclei overnight in the presence of 0.02% digitonin at 4 degrees. The next day, 700ng/mL of proteinA-micrococcal nuclease (pA-Mnase purified in house with vector from Addgene 86973, protocol from Schmid et al.) (Schmid et al., 2004) were incubated with the nuclei at 4 degrees for an hour. After washing, the tubes were placed in heat blocks on ice set to 0 degrees, CaCl2 (1mM) was added and incubated for 30 minutes before 2x Stop buffer containing EDTA was added. DNA was eluted by heat and high-speed spin, then phenol-chloroform extracted. Qubit was used to quantify purified DNA and Bioanalyzer (2100) traces were run to determine the size of the cleaved products. NEBNext Ultra II DNA library prep kit (NEB E7645) was used to make the libraries according to Liu et al.'s protocol, outlined on protocols.io (dx.doi.org/10.17504/protocols.io.wvgfe3w) (Liu et al., 2018b) . Pair-end (42bp) Illumina sequencing was performed on the bar-coded and amplified libraries.
Kong N.R., Bassal M.A., Tan H.K., Kurland J.V., Yong K.J., Young J.J., Yang Y., Li F., Lee J., Liu Y., Wu C., Stein A., Luo H.R., Silberstein L.E., Bulyk M.L., Tenen D.G., & Chai L. (2020). Zinc finger protein SALL4 functions through an AT-rich motif to regulate gene expression.
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