ChIP analysis was performed as reported previously (14 (link)) by using 5 μg rabbit α-SNAIL (R&D system), α-EZH2 (Active motif) or the negative control normal rabbit immunoglobulin (IgG) (Millipore Corp., Bedford, MA) or normal goat immunoglobulin (IgG) (R&D system). 5 ng of immunoprecipitated DNA and the relative controls were used as templates for real-time qPCR analysis, performed in triplicate. qPCR analysis of the immunoprecipitated samples and of the negative controls (IgG) were both normalized to total chromatin input and expressed as percentage of Input (% Input). Histone ChIP analysis was performed by using 5 μg of the specific antibody (H3K27me3; 07-449; Millipore Corp., Bedford, MA) or of the negative control normal rabbit IgG (Millipore Corp., Bedford, MA), as reported previously (14 (link)). The DNA was extracted with phenol-chloroform, precipitated with ethanol and resuspended in 50μl of water, then used in the downstream qPCR analyses (primer pairs details are listed in Table S2 ).
H3k27me3 07 449
Manufactured by Merck Group
Sourced in United Kingdom
H3K27me3 (07-449) is a laboratory reagent used to detect the presence of trimethylated histone H3 at lysine 27 (H3K27me3), a post-translational modification associated with transcriptional repression. This product is intended for research use only and its specific applications should be evaluated by the user.
Automatically generated - may contain errors
Lab products found in correlation
Protein transport inhibitor, by Thermo Fisher Scientific (2 mentions)
H3k27ac ab4729, by Abcam (2 mentions)
Foxp3 transcription factor staining buffer kit, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Normal rabbit igg, by Merck Group (1 mentions)
Rbbp5, by Fortis Life Sciences (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Ab1791, by Abcam (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Xm10 digital camera, by Olympus (1 mentions)
Hpa053669, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Boster Bio (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ix51 fluorescence microscope, by Olympus (1 mentions)
5 protocols using h3k27me3 07 449
1
ChIP-qPCR Analysis of Chromatin Modifications
2
Multi-parameter Cytokine and Transcription Factor Profiling
3
ChIP-seq Analysis of Chromatin Regulators
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ChIP was performed as previously described [56 (link)]. For CFP1, HCF1, RbBP5, hSET1 (SETD1A), Menin and UTX, chromatin was first cross-linked with ethylene glycol bis(succinimidyl succinate) (EGS) [29 (link)] in PBS at a final concentration of 2 mM for 60 min at RT. Formaldehyde (CH2O) was then added at a final concentration of 1% for 15 min at RT and samples were sonicated over 20 min (10 × 30-second episodes) at 4 °C to cleave genomic DNA (Bioruptor, Diagenode). Input data sets were matched with ChIP-seq samples. Antibodies used are: H3K27me3 (07-449) (Millipore); HCF1 (A301-400A), RbBP5 (A300-109A), hSET1 (SETD1A, A300-289A), Menin (A300-105A), UTX (A302-374A) from Bethyl Labs and CFP1 (CGBP, ab56035) from Abcam. The non-commercial CFP1 antibody was kindly provided by Prof. Robert Roeder. Real-Time PCR was performed using primers and probes (5’FAM-3’TAMRA) for the murine and human α-globin locus described previously [57 (link), 58 (link)]. Each ChIP was performed as two independent experiments and quality was assessed by qPCR. Libraries and sequencing (ChIP-seq) were performed using the standard Illumina kits and protocols.
4
Multi-parameter Cytokine and Transcription Factor Profiling
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KLRG1 (2F1), CD127 (A7R34), CD8 (53-6.7), CD45.1 (A20-1.7), CD45.2 (104), CXCR3 (CXCR3-173), CD27 (LG-7F9), T-bet (4B10), Bcl-6 (K112-91), were purchased from eBioscience. FOXO1 (C29H4), TCF-1 (C63D9), IFN-γ (XMG1.2), TNF (MP6-XT22) were from Cell Signaling Technology. Antibodies for ChIP-seq, H3K4me3 (Ab8580), H3K4me1 (Ab8895) and H3K27ac (Ab4729) were from Abcam. H3K27me3 (07-449) was from Millipore. For intracellular staining of cytokines, splenocytes were in vitro restimulated with 1 μg/ml OVA peptide (SIINFEKL) with Protein Transport Inhibitor (eBioscience) for 4 h and then fixed and permeabilized using BD cytofix/cytoperm kit (BD Biosciences). Foxp3-transcription factor staining buffer kit (eBioscience) were used for intracellular staining of transcription factors. For intracellular staining of shRNA-transduced cells containing Ametrine-reporter, cells were fixed using freshly made 2% formaldehyde for 45 min on ice and then permeabilized. All flow cytometry data were acquired by BD LSRFortessa X-20 and all cell sorting was performed on BD FACS Aria.
5
Analyzing Histone H3 Methylation and SIRT7 Interaction
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Sh-scramble/CTRL and sh-SIRT7 cells, were seeded in 1 cm2 coverslips and allowed to grow overnight. Then, cells were fixed in 4% formaldehyde (Sigma) for 10 min and permeabilized in 0.5% Triton X-100 (Sigma), for 5 min, at RT and gently stirred. PLA assay was performed using the commercial kit Duolink In Situ (OLINK Bioscience, Uppsala, Sweden), according to manufacturer’s instructions. The antibodies used were Histone H3 (ab1791, Abcam, Cambridge, UK), tri-methylation of Lysine 27 of Histone H3 (H3K27me3, 07-449, Millipore), SIRT7 (HPA053669, Sigma-Aldrich) and EZH2 (NCL-L-EZH2, Leica Biosystems). After the procedure, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (AR1176, BOSTER Biological Technology, Pleasanton, CA, USA) in mounting medium. Pictures were taken on an Olympus IX51 fluorescence microscope equipped with an Olympus XM10 digital camera using CellSens software.
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