Ab4729

Manufactured by Merck Group

Ab4729 is a laboratory equipment product manufactured by Merck Group. It is designed for use in scientific research and analysis. The core function of Ab4729 is to facilitate the detection and quantification of specific biomolecules, such as proteins or nucleic acids, in a sample. Further details about the intended use or specific applications of this product are not available.

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Lab products found in correlation

15 protocols using ab4729

Profiling Chromatin Features of Bidirectional Gene Pairs

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ChIP-seq datasets (GSE79033) [32 (link)] for H3K4ac (Millipore, 07–539), H3K9ac (Millipore, 07–352), H3K27ac (Abcam,ab4729), H3K27me3 (Millipore, 07–449), H3K9me1 (Millipore, 07–395) and H3K9me3 (Millipore, 07–442), were generated from seeding using a previously described method [44 (link)]. Six of previously characterized ChIP-seq and MNase-seq (SRP045236)⁴⁶ datasets obtained from seedlings for H3K4me3, H3K4me2, H3K36me3 and K4K12ac (GSE26734) [44 (link)] and H4K16ac and H3K23ac (GSE69426) [45 (link)] were downloaded from NCBI for further analysis. All ChIP-seq datasets were analyzed using the same pipeline as previously described [44 (link)].
To confirm the ChIP-seq results, we conducted a ChIP-qPCR assay following ChIP experiments using two histone marks (H3K27ac and H4K12ac). Five of the BDPs were randomly selected to design primers (Additional file 15: Table S11) for ChIP-qPCR analysis. One primer set was triplicated in the qPCR assay.
To profile the chromatin features of histone marks and nucleosome positioning associated with bidirectional gene pairs, we plotted the normalized ChIP-seq and MNase-seq reads across all bidirectional gene pairs and randomly selected unidirectional genes as controls.

Fang Y., Wang L., Wang X., You Q., Pan X., Xiao J., Wang X.E., Wu Y., Su Z, & Zhang W. (2016). Histone modifications facilitate the coexpression of bidirectional promoters in rice. BMC Genomics, 17, 768.

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ChIP Assay for Histone Modifications

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ChIP was conducted essentially as previously described with 150 embryos per condition [131 (link)]. Antibodies used for IPs were: anti-GFP (Abcam; ab290), HDAC1 (Abcam; ab41407), H3K27ac (Abcam; ab4729), and H3K27me3 (Millipore; 7449). All IPs were performed using 1:100 dilutions of the antibodies. The IP’d DNA was analyzed with qPCR described above. Fold enrichment of ripply3-DR1 and ripply3-DR4 were standardized to that of negative control samples (IgG) (Southern Biotech; 617001). Primer sequences for IPs are listed in S1 Table.

Song Y.C., Dohn T.E., Rydeen A.B., Nechiporuk A.V, & Waxman J.S. (2019). HDAC1-mediated repression of the retinoic acid-responsive gene ripply3 promotes second heart field development. PLoS Genetics, 15(5), e1008165.

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ChIP-qPCR and ChIP-seq for Epigenomic Profiling

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ChIP followed by quantitative polymerase chain reaction (ChIP-qPCR) was performed as described previously (31 (link)). Antibodies specific for CTCF (Millipore 07–729) or normal rabbit IgG (Millipore 12-370) were used. qPCR was performed using SYBR Green reagents (Life Technologies) and primers listed in Supplementary Table S1B.
ChIP and deep-sequencing (ChIP-seq) data was generated according to standard protocol as described previously (20 (link)). Antibodies used were from Abcam (ab) or Millipore (mi) and specific for H3K4me1 (ab8895), H3K27ac (ab4729), H3K4me3 (mi07–473), H3K27me3 (mi07–449), H3K9me2 (ab1220), H3K9me3 (ab8898) and H3K36me3 (ab9050). H3K4me1 and H3K27ac data in Calu3 and HBE cells used here were generated previously (Calu3 (20 (link)), HBE (55 )) All data are deposited at GEO (http://www.ncbi.nlm.nih.gov/geo; GSE63400, GSE74709, GSE94726).

Stolzenburg L.R., Yang R., Kerschner J.L., Fossum S., Xu M., Hoffmann A., Lamar K.M., Ghosh S., Wachtel S., Leir S.H, & Harris A. (2017). Regulatory dynamics of 11p13 suggest a role for EHF in modifying CF lung disease severity. Nucleic Acids Research, 45(15), 8773-8784.

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Chromatin Immunoprecipitation Sequencing

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Cells (10⁷) were cross-linked with 1% formaldehyde (EMD Millipore, USA, 344198) at room temperature for 10 min. The chromatin was obtained by cell lysis (50 mM HEPES, pH 7.5; 150 mM NaCl; 1mM EDTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; and proteinase inhibitors) and was followed by sonication (Sonics VCX130, USA) on ice for 6 min. ChIP-grade antibodies (10 μg), anti-H3K44me3 (Abcam, ab8580), anti-H3K27ac (Abcam, ab4729), anti-H3K27me3 (Millipore, 07-499), and anti-RNA polymerase II (Abcam, ab817), were incubated with 100 μL Protein G DynaBeads (Life, 10009D) at 4 °C for 2 h. The chromatin and the antibody coated beads were co-immunoprecipitated on a rotator at 4 °C overnight. The ChIP DNA was de-crosslinked by protein K (Ambion, AM2546) digestion at 55 °C overnight. DNA (1 μg) was used for the preparation of the Chromatin Immunoprecipitation Sequencing (ChIP-Seq) library by NEBNext UltraⅡ DNA Library Prep Kit for Illumina (NEB, USA, E7645 and E7335), according to the manufacturer’s instructions. Each ChIP-Seq library was high-throughput sequenced, ~50M paired-end reads (2 × 150 bp) on Illumina HiSeq3000 platform.

Wang S., Sun Y., Ren R., Xie J., Tian X., Zhao S., Li X, & Cao J. (2019). H3K27me3 Depletion during Differentiation Promotes Myogenic Transcription in Porcine Satellite Cells. Genes, 10(3), 231.

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H3K27Ac ChIP-qPCR Profiling of p21 Locus

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FACS-enriched Tom⁺ cell suspensions from Ai14;L-KRAS^G12V or Ai14 control livers (see below) were pelleted, resuspended in medium, and counted. A total of 1–4×10⁵ cells were fixed with 1% PFA for 10 min and then subjected to H3K27Ac-ChIP using a rabbit anti-H3K27Ac antibody (Abcam, ab4729, Lot GR150367) or rabbit, IgG (Millipore, #12–370) according to the manufacturers protocol (Active Motif, #53084). Precipitated chromatin or input DNA was subjected to quantitative PCR in the indicated genomic regions in the SASE of the p21 locus using primers indicated in table S9.

Sturmlechner I., Zhang C., Sine C.C., van Deursen E.J., Jeganathan K.B., Hamada N., Grasic J., Friedman D., Stutchman J.T., Can I., Hamada M., Lim D.Y., Lee J.H., Ordog T., Laberge R.M., Shapiro V., Baker D.J., Li H, & van Deursen J.M. (2021). p21 produces a bioactive secretome that places stressed cells under immunosurveillance. Science (New York, N.Y.), 374(6567), eabb3420.

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Integrative Genomic Analysis of CTCF Regulation

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ATAC-seq, CTCF ChIP-seq, and Hi-C datasets as well as allelic data analyses are described in our previous studies [8 (link),15 (link),27 (link)]. Virtual 4C analysis was performed as described [15 (link)]. ChIP-seq for RAD21 was performed in WT and CTCF-site edited Patski cells using a rabbit monoclonal antibody for RAD21 (Abcam ab217678) as described previously [8 (link)]. CUT&RUN was performed in WT and CTCF-site edited Patski cells using antibodies for CTCF (Millipore 07-729), H3K27ac (Abcam ab4729) and H3K27me3 (Millipore 07-449), as described in [27 (link)]. Allelic data analysis was done as described [15 (link)]. Additional CUT&RUN datasets of CTCF, H3K4me3, H3K27ac3, and H3K36me3 in WT and Firre-deleted Patski cells were obtained from Thakur et al. [40 ]. Note that no allelic analyses were possible due in this batch to limited SNP coverage in short (36nt) reads.

Fang H., Tronco A.R., Bonora G., Nguyen T., Thakur J., Berletch J.B., Filippova G.N., Henikoff S., Shendure J., Noble W.S., Disteche C.M, & Deng X. (2023). CTCF-mediated insulation and chromatin environment modulate Car5b escape from X inactivation. bioRxiv.

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Histone ChIP-seq Analysis in Embryonic Tissue

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ChIP-seq data sets for four histone marks (H3K4me1, H3K4me3, H3K27ac, and H3K27me3—Abcam ab8895, ab8580, ab4729, and Millipore 07-449, respectively) were generated for E11.5 limbs, while for E11.5 forebrain we produced ChIP-seq for H3K4me1, H3K4me3, and H3K27ac and used a previously generated H3K27ac data set (Nord et al. 2013 (link)). Histone ChIP-seq was carried out using previously described protocols (Bernstein et al. 2006 (link)). DNA libraries were prepared, sequenced, and analyzed as described for FLAG ChIP-seq. All histone data sets were generated with an input control.

Attanasio C., Nord A.S., Zhu Y., Blow M.J., Biddie S.C., Mendenhall E.M., Dixon J., Wright C., Hosseini R., Akiyama J.A., Holt A., Plajzer-Frick I., Shoukry M., Afzal V., Ren B., Bernstein B.E., Rubin E.M., Visel A, & Pennacchio L.A. (2014). Tissue-specific SMARCA4 binding at active and repressed regulatory elements during embryogenesis. Genome Research, 24(6), 920-929.

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ChIP-seq analysis of transcription factors

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ChIP was performed using standard methods as previously described (1 (link),8 (link)). Antibodies were against FOXA1 (Abcam ab5089), CDX2 (Bethyl Laboratories A300–691A), FOXA2 (SCB sc-6554x), histone H3 (ab1791), H3K9Me3 (ab8898), H3K9Ac (Millipore 07–352), H3K27ac (ab4729), CTCF (Millipore 07–729), RAD21 (ab992), SMC1 (Bethyl Laboratories A300–055A), normal goat IgG (SCB sc-2028), or normal rabbit IgG (Millipore 12–370). qPCR was performed using SYBR® Green reagents with primers listed in Supplementary Table S1.

Gosalia N., Neems D., Kerschner J.L., Kosak S.T, & Harris A. (2014). Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus. Nucleic Acids Research, 42(15), 9612-9622.

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Chromatin Immunoprecipitation and Immunoblotting Protocol

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Rabbit monoclonal anti-UTX, Cell Signaling 33510, vendor information indicates that the immunogen is a recombinant protein surrounding Ala490 of human UTX, and our data confirmed that it reacts with the UTX region between 419 and 548. Rabbit polyclonal anti-MLL4, Sigma HPA035977 (immunoblotting); Santa Cruz Biotec. sc-293217 (ChIP); Rabbit polyclonal anti-MLL3, a gift from Kai Ge; Mouse monoclonal anti-GAPDH, Millipore MAB374; Rabbit monoclonal anti-H3K4me3, Millipore 05-745R; Rabbit polyclonal anti-H3K27me3, Millipore 07-449; anti-H3K27ac, Abcam ab4729; anti-H3K4me2, Millipore 07-030; anti-H3K4me1, Abcam ab8895; Rabbit polyclonal anti-RBBP5 and -WDR5, Bethyl Laboratory A300-109A and A302-429A, respectively; Mouse monoclonal anti-c-Myc (9E10), ThermoFisher Scientific, 132500; Mouse monoclonal anti-FLAG M2 Affinity agarose, Sigma, A2220; Alexa Fluor 555 conjugated goat anti-rabbit IgG, ThermoFisher Scientific A-21428; Alexa Fluor 488 conjugated goat anti-mouse IgG, ThermoFisher Scientific A-11001.

Shi B., Li W., Song Y., Wang Z., Ju R., Ulman A., Hu J., Palomba F., Zhao Y., Le J.P., Jarrard W., Dimoff D., Digman M.A., Gratton E., Zang C, & Jiang H. (2021). UTX condensation underlies its tumor-suppressive activity. Nature, 597(7878), 726-731.

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ChIP-seq and qPCR Protocol for Epigenomic Analysis

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For ChIP followed by qPCR, 2–5 million primary keratinocytes were used as starting material; for ChIP–seq, 10–30 million cells were used. ChIP was performed essentially as described^{24 (link)}. Pulldown was performed with 10 µg of ChIP–seq-validated antibody against H3K27ac (Abcam, ab4729), CTCF (Millipore, 07-729), SMC1A (Bethyl, A300-055A), or EHF (Santa Cruz Biotechnology, sc-166653). Staph A cells were used for pulldown. DNA was purified using Qiagen QIAquick PCR Purification columns and subjected to qPCR (primer sequences in Supplementary Table 7). For qPCR, the percentage of input signal was calculated, and error bars represent s.e.m. calculated using GraphPad Prism. ChIP–seq libraries were prepared with the NEBNext ChIP–seq library prep kit (NEB) using AMPure beads (Agencourt) for purification.

Rubin A.J., Barajas B.C., Furlan-Magaril M., Lopez-Pajares V., Mumbach M.R., Howard I., Kim D.S., Boxer L.D., Cairns J., Spivakov M., Wingett S.W., Shi M., Zhao Z., Greenleaf W.J., Kundaje A., Snyder M., Chang H.Y., Fraser P, & Khavari P.A. (2017). Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation. Nature genetics, 49(10), 1522-1528.

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