Mouse SETDB1 iKO-ES cells were treated with Tam for 3 d. Cells were then treated with fresh culture medium containing 1% formaldehyde for 10 min and washed twice with ice-cold PBS containing protease inhibitors. Cell pellets were resuspended in SDS lysis buffer also containing protease inhibitors (200 µL lysis buffer for every 1 × 106 cells) and incubated on ice for 10 min. Cell lysate was sonicated (15 W for 10 sec, six times) to shear DNA to lengths between 100 and 500 bp. Subsequently, ChIP was performed according to the ChIP assay kit (Millipore17-295) instructions using antibodies against SETDB1 (Santa Cruz, no. 66884), EZH2 (Millipore, no. 17-662), H3K27me3 (Millipore, no. 07-449), and H3K9me3 (Active Motif, no. 39161). Eluted DNA was used for PCR, qPCR, or deep sequencing. For ChIP-qPCR analysis, the relative binding level of each gene was normalized against input. Primer sequences are listed in Supplemental Table S2. For ChIP-seq libraries, 10 ng of input chromatin DNA or ChIP DNA was processed using the ChIP-seq sample prep kit (Illumina). Gel-purified ChIP-seq library DNA was further purified by phenol-chloroform extraction and ethanol precipitation and was processed for cluster generation, 15-cycle sequencing, and sequence analysis using Illumina HiSeq. The summary of generated ChIP-seq data sets is listed in Supplemental Table S3.
Setdb1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
SETDB1 is a histone methyltransferase that catalyzes the trimethylation of lysine 9 on histone H3 (H3K9me3). This epigenetic modification is associated with transcriptional repression and heterochromatin formation.
Automatically generated - may contain errors
Lab products found in correlation
H3k27me3, by Merck Group (3 mentions)
H3k9ac, by Merck Group (2 mentions)
H3k4me3, by Merck Group (2 mentions)
H3k9me3, by Abcam (2 mentions)
H3k9me3, by Merck Group (2 mentions)
H3k9me3, by Active Motif (1 mentions)
Chip seq sample prep kit, by Illumina (1 mentions)
Chip assay kit, by Merck Group (1 mentions)
Ab8898, by Abcam (1 mentions)
Rif 1, by Santa Cruz Biotechnology (1 mentions)
Suz12, by Cell Signaling Technology (1 mentions)
Nuclear complex co ip kit, by Active Motif (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Chemismart 5000 system, by Vilber (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by CWBIO (1 mentions)
Gfra1, by Santa Cruz Biotechnology (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Mayer hematoxylin solution, by Merck Group (1 mentions)
7 protocols using setdb1
1
ChIP-seq Analysis of Setdb1 in Mouse ES Cells
2
Profiling Epigenetic Marks in Cell Lines
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Antibodies used in this study include: HA (C29F4, CST 3724), RIF1 (Santa Cruz SC-65191), EZH2 (CST 5246), SETDB1 (Santa Cruz sc-66884), H3K9me3 (Abcam ab8898), H3K4me3 (Active motif 39159), H3K27me3 (Active motif 39155), H3K9ac (Millipore 07-352), H3K27ac (Active motif 31933), Suv39H1 (CST 8729), EHMT2 (CST 3306).
3
Epigenetic Modification Analysis by Antibody Staining
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Antibodies used in this study include: HA (C29F4, CST 3724), RIF1 (Santa Cruz, SC-65,191), EZH2 (CST, 5246), SETDB1 (Santa Cruz, sc-66,884), H3K9me3 (Abcam, ab8898), H3K4me3 (Active motif, 39,159), H3K27me3 (Active motif, 39,155), H3K9ac (Millipore, 07-352), H3K27ac (Active motif, 31,933), Suv39H1 (CST, 8729), EHMT2 (CST, 3306).
4
Nuclear Protein Complex Co-IP
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Co-IP experimental procedures were performed following the protocol of nuclear complex co-IP kit (Active Motif, no. 54001). The nuclear complex was collected for IP using antibodies against SETDB1 (Santa Cruz, no. 66884), EZH2 (Cell Signaling, no. 3147), and SUZ12 (Cell Signaling, no. 3737). Post-IP protein G beads were washed three times with 1× wash buffer, and proteins were eluted with 2× SDS-loading buffer. Samples were then incubated for 10 min at 99°C before being loaded for SDS-PAGE.
5
Western Blot Analysis of SETDB1 and MPP8
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Nuclear extracts or immunoprecipitates were resolved on pre-cast polyacrylamide gel cassettes (NuPAGE® Novex® 4–12% Bis-Tris) (Invitrogen; Waltham, Massachusetts, USA) and 1X NuPAGE MES SDS Running Buffer and transferred into nitrocellulose membrane (Amersham; Chicago, Illinois; USA) in 20 mM phosphate transfer buffer (pH 6.7). Membrane was blocked in 5% skim milk in PBST Buffer (1X PBS, 0.2% Tween 20) and incubated overnight at 4 °C with the primary antibodies against SETDB1 (Cat# sc-66884; Santa Cruz Biotechnologies; Dallas, Texas; USA), MPP8 (Cat# 16796-1-AP; Proteintech; Rosemont, Illinois; USA). Membranes were incubated with the appropriate secondary antibody coupled to HRP, revealed using West Dura kit (Pierce, Rockford, USA) and ChemiSmart 5000 system (Vilber Lourmat; Marne-la-Vallée; France). When necessary, the TrueBlot secondary Ab (Clinisciences; Nanterre; France) was used to reduce interference with the ~55 kDa heavy and ~23 kDa light chains of immunoprecipitating IgG.
6
Immunofluorescence Staining of GFRA1, SETDB1, H3K9me3, and c-KIT
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The procedure was similar to that in immunohistochemistry section with minor modification. Briefly, the slides were incubated with 10% donkey serum for blocking nonspecific reactions at room temperature for 2 h and incubated with the primary antibodies against GFRA1 (1:50; Santa Cruz Biotechnology), SETDB1 (1:50; Santa Cruz Biotechnology), H3K9me3 (1:50; Millipore) and c-KIT (1:50; Santa Cruz Biotechnology) respectively, overnight at 4°C. Next day, the sections were washed four times with PBS and then incubated with either donkey anti-Goat (1:200; Abcam) or donkey anti-Rabbit (1:100; Santa Cruz Biotechnology) antibodies for immunofluorescence labeling. Slides were washed and counterstained with DAPI (CWBIO). Fluorescent images were captured with the Nikon Eclipse 80i fluorescence microscope camera.
7
Immunohistochemical Analysis of SETDB1 and H3K9me3
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The 6-μm-thick sections were de-paraffinized with xylene and re-hydrated with ethanol series from 100% (v/v) to 70% (v/v). The slides were washed three times, each for 5 min in PBS, followed by 0.5% Triton X-100 in ultrapure water for 10 min at room temperature. The slides were washed and boiled in a solution of 0.01-M Tris-EDTA (pH = 9) for 15 min for antigen retrieval, and cooled at room temperature for 40 min. The sections were incubated with 3% H 2 O 2 to block endogenous peroxidase, washed three times with PBS, and blocked with 10% goat serum for 2 h at room temperature. Subsequently, the slides were incubated with the primary antibodies against SETDB1 (1:50; Santa Cruz Biotechnology), or H3K9me3 (1:50; Millipore) at 4°C overnight. While some slides were incubated with blocking solution as a negative control. Next day, the slides were rinsed four times in PBS, incubated with biotinylated secondary antibody for 1 h at 37°C and then washed three times with PBS. The slides were exposed to the horse radish peroxidase at 37°C for 1 h, followed by incubation in 3,3′-diaminobenzidine (CWBIO, Beijing, China) for staining and counterstained with Mayer hematoxylin solution (Sigma-Aldrich), and washed three times with PBS for 5 min each. Digital images were captured with the Nikon Eclipse 80i microscope camera.
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