The assay was performed as recently described47 (link). Briefly, HepG2 cells were seeded in a 60-mm dish, and the following day, they were incubated with carvones (1000 μM) in combination with vehicle (0.1% DMSO) or the AhR agonists TCDD (20 nM), BaP (7 μM), and FICZ (8 nM) for 90 min at 37 °C. The procedure followed the manufacturer’s recommendations for the SimpleChIP Plus Enzymatic Chromatin IP kit (Magnetic Beads) (Cell Signaling Technology; #9005). Anti-AhR rabbit monoclonal antibody was purchased from Cell Signaling Technology (D5S6H; #83200). CYP1A1 promoter primers were (fw: AGCTAGGCCATGCCAAAT, rev: AAGGGTCTAGGTCTGCGTGT-3’). Experiments were performed in three consecutive cell passages. Full scan gels are available at 10.5281/zenodo.7764002.
Simplechip plus enzymatic chromatin ip kit magnetic beads
Manufactured by Cell Signaling Technology
Sourced in United States
The SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) is a laboratory tool designed for the analysis of protein-DNA interactions through chromatin immunoprecipitation (ChIP) experiments. The kit provides reagents and protocols to efficiently extract and fragment chromatin from cells, perform immunoprecipitation using magnetic beads, and purify the DNA for downstream analysis.
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Lab products found in correlation
D5s6h, by Cell Signaling Technology (1 mentions)
Tgf beta 1, by Thermo Fisher Scientific (1 mentions)
Opti mem reduced serum media, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Pierce crosslink magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Messengermax, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 ultra transcription kit, by Thermo Fisher Scientific (1 mentions)
Genelute pcr clean up kit, by Merck Group (1 mentions)
H3k4me3 antibody, by Cell Signaling Technology (1 mentions)
7 protocols using simplechip plus enzymatic chromatin ip kit magnetic beads
1
AhR Binding and CYP1A1 Regulation in HepG2 Cells
2
Chromatin Immunoprecipitation and RNA Interference Protocols
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Antibodies information can be found in Supplementary Table S1 . Lipofectamine® 3000, RNAiMAX, MessengerMAX, Opti-MEM® reduced serum media, and SYBR® Select Master Mix, were obtained from Invitrogen® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA). RPMI-1640, DMEM medium, and FBS were obtained from Gibco® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA). PrimeScript® RT reagent Kit with gDNA Eraser was obtained from Takara bio (Dalian, China). TGF-Beta1 was purchased from PeproTech® (London, UK). mMESSAGE mMACHINE® T7 ULTRA Transcription Kit was obtained from Ambion® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA) LY2109761(Cat.#S2704) was obtained from Selleck (Houston, TX, USA)
SimpleChIP® Plus Enzymatic Chromatin IP Kit-Magnetic Beads (#9005) were purchased from Cell Signaling Technology (Danvers, MA, USA), and Pierce® Crosslink Magnetic IP/Co-IP Kit(#88805) came from Thermo Fisher Scientific (Waltham, MA, USA). siRNAs for RbBP5, SNAI1, CBP, SMAD2 were synthesized by Ribo Bio(Guangzhou, China). For their sequences, see theSupplementary Table S2 .
SimpleChIP® Plus Enzymatic Chromatin IP Kit-Magnetic Beads (#9005) were purchased from Cell Signaling Technology (Danvers, MA, USA), and Pierce® Crosslink Magnetic IP/Co-IP Kit(#88805) came from Thermo Fisher Scientific (Waltham, MA, USA). siRNAs for RbBP5, SNAI1, CBP, SMAD2 were synthesized by Ribo Bio(Guangzhou, China). For their sequences, see the
3
Enzymatic ChIP-seq Protocol for Cell Lines
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SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 from Cell Signaling Technologies was used for all ChIP-seq experiments using manufacturer's recommended protocol. Four million cells per IP. Digested chromatin was pooled into single tube for brief sonication to lyse nuclei. Supernatant was then split evenly between IPs (minus 2% input). Antibodies and chromatin were incubated overnight at 4°C, rotating. DNA was purified using spin columns and prepared using NEB Ultra II DNA library kit.
4
Hypoxia-Induced β-Catenin ChIP Assay
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Cells treated with 1% oxygen and Simple ChIP plus enzymatic chromatin IP kit (magnetic beads) #9005 (Cell Signaling Technology, Beverly, Massachusetts, USA) was used as described in manufacturer’s instructions. Briefly, cells were fixed with formaldehyde to cross-link histone and non-histone proteins to DNA. The chromatin was digested with micrococcal nuclease into 150-900 bp DNA/protein fragments. Anti-β-Catenin antibodies was added followed by incubation overnight at 4°C and was captured by protein G magnetic beads. The protein/DNA complex was disintegrated, and DNA was purified for analysis. High-throughput sequencing was performed by Shanghai Outdo Biotech Co., Ltd.
5
Chromatin Immunoprecipitation (ChIP) from Tissue
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ChIP using tissue was performed according to the manufacturer's instructions (Abcam). Briefly, ~20 mg frozen EPN tissue was minced into 1–3 mm3 pieces, thawed on ice, washed with ice cold PBS with proteinase inhibitor cocktail prior to fixation in 1% formaldehyde in PBS at room temperature for 10 min and quenched with 0.125 M glycine for 5 min. Tissue was collected and resuspended in 500 μl FA-lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL pepstatin) and disrupted while on ice, with a Wheaton Dounce Tissue Grinder (VWR International, 62,400–595). The tissue suspension was transferred into a 5 ml conical tube, sonicated and processed according to manufacturer's protocol using rabbit polyclonal H3K4me3 antibody (Cell Signaling Technology, Cat# 9727) and IgG from rabbit serum (Cell Signaling Technology, Cat# 2729) to collect immunoprecipitated DNA fragments. All DNA fragments were cleaned-up with GenElute PCR clean-Up Kit (Sigma Aldrich Co., St. Louis, MO, USA) prior to PCR analysis. ChIP using cells was performed with the SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic beads) (Cell Signaling Technology, Cat#9005) following manufacturer's instructions. Human ERBB2 and Cyclin D1 ChIP primers (Supplementary Table 3) were used following real-time PCR analysis.
6
ChIP-seq Assay Using Enzymatic Protocol
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The SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, Danvers, MA, USA; #9005S) was used to perform the ChIP-seq experiment. Cells were fixed in 1% formaldehyde, harvested, sonicated, and then incubated with anti-acetyl-histone H3 (AcH3; Upstate Biotechnology, Los Altos, CA, USA; 06-599), anti-RNA polymerase II (RNA pol II; Covance, MMS-126R), or IgG antibody (Cell Signaling Technology, #2729). Purified ChIP DNA was confirmed by PCR using primers for the UPP1 promoter (F, 5’-GG CTTGTCTGCGGGATG-3’ and R, 5’-CGGAGCACTC GAATGAGG-3’).
7
Enzymatic Chromatin Immunoprecipitation
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SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 from Cell Signaling Technologies was used for all ChIP-seq experiments using the manufacturer's recommended protocol. Four million cells per IP. Digested chromatin was pooled into a single tube for brief sonication to lyse nuclei. Supernatant was then split evenly between IPs (minus 2% input). Antibodies and chromatin were incubated overnight at 4°C, rotating. DNA was purified using spin columns and prepared using NEB Ultra II DNA library kit.
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