ChIP-seq assays were performed on the fourth leaves of 4-week-old tomato plantsaccording to Bio-protocol of Ramirez-Prado et al.69 (link) using 3ug of anti-H3K27me1 (Millipore, 07–448), anti-H3K9ac (Millipore, 07–352), anti-H3K14ac (Millipore, 07–353), anti-H3K18ac (Millipore, 07–354), anti-H3K27ac (Abcam, ab4729), anti-H3K4me3 (Millipore, 07–473), and anti-RNAPII (Abcam, ab26721) antibodies. ChIP-seq libraries were prepared from 10 ng of DNA using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions. Two independent biological replicates were generated for each time point of heat stress. DNA libraries were checked for quality and quantified using a 2100 Bioanalyzer (Agilent) and subjected to 1 × 75 bp high-throughput sequencing by NextSeq 500 (Illumina).
Anti h3k27me1
Manufactured by Merck Group
Anti-H3K27me1 is a laboratory reagent used to detect and quantify the presence of the histone modification H3K27me1 in various biological samples. It is a specific antibody that binds to the monomethylated form of lysine 27 on histone H3 proteins. This reagent can be used in techniques such as Western blotting, immunohistochemistry, and chromatin immunoprecipitation to analyze the epigenetic landscape of cells and tissues.
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Lab products found in correlation
Anti h3k4me3, by Merck Group (4 mentions)
Anti h3k27me3, by Merck Group (4 mentions)
Anti h3k27me2, by Merck Group (3 mentions)
Anti h3k4me2, by Merck Group (3 mentions)
Anti h3k4me1, by Merck Group (3 mentions)
Anti eed, by Merck Group (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti h3k9me2, by Merck Group (2 mentions)
Anti h3k9me1, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Anti h3k9ac, by Merck Group (1 mentions)
Anti h3k18ac, by Merck Group (1 mentions)
Anti h3k14ac, by Merck Group (1 mentions)
Nebnext ultra 2 dna library prep kit for, by Illumina (1 mentions)
Anti h3k27ac, by Abcam (1 mentions)
Nextseq 500, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Anti suz12, by Cell Signaling Technology (1 mentions)
Anti histone 3, by Merck Group (1 mentions)
Anti p16, by Santa Cruz Biotechnology (1 mentions)
9 protocols using anti h3k27me1
1
ChIP-seq analysis of histone marks in tomato
2
Comprehensive Protein Expression Analysis
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Lysates were prepared in RIPA buffer [16 (link)], and western blotting was performed as described [17 (link)]. The following antibodies were used for western blot analysis: anti-Ezh2 (BD Biosciences #612666), anti-Suz12 (Cell Signaling 3737S), anti-Eed (Millipore 05–1320), anti-H3K27me1 (Millipore 07–448), anti-H3K27me2 (Millipore 07–452), anti-H3K27me3 (Millipore 07–449), anti-Histone 3 (Millipore 07–690), anti-β-actin (Sigma A5441), anti-Gapdh (Sigma G8795), anti-ERα (Millipore 07–690), anti-Ezh1 (Millipore 07–690), anti-PARP (Cell Signaling #9542), anti-p16 (Santa Cruz M-156), and anti-p19 (Rockland Immunochemicals #200-501-891). Secondary antibodies included HRP-conjugated anti-rabbit and anti-mouse (Southern Biotech), both 1:10,000.
3
Histone Modification and Signaling Pathway Analysis
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Cells were harvested and lysed in RIPA buffer with protease inhibitors and 2 μM PMSF. Protein extracts were boiled in SDS sample buffer for 5 min, loaded directly onto a 4–12% SDS gel, transferred onto nitrocellulose membranes (Bio-Rad), blocked with 5% milk, and incubated with corresponding primary and secondary antibodies using standard protocols. The following antibodies were used: anti-H3K4me1 (Cat # 07-436, 1:2000 dilution), anti-H3K4me2 (Cat # 07-030, 1:4000 dilution), anti-H3K4me3 (Cat # 07-473, 1:4000 dilution), anti-H3K9me1 (Cat # 07-450, 1:2000 dilution), anti-H3K9me2 (Cat # 07-441, 1:3000 dilution), anti-H3K9me3 (Cat # 07-442, 1:3000 dilution), anti-H3K27me1 (Cat # 07-448, 1:2000 dilution), anti-H3K27me2 (Cat # 07-452, 1:4000 dilution), and anti-H3K27me3 (Cat # 07-449, 1:4000 dilution) from Millipore; rabbit anti-Jmjd3 (Cat # ab1022a, 1:500 dilution) from Abgent; and mouse anti-FLAG (1:5000 dilution), anti-HRP-FLAG (1:5000 dilution), and anti-β-actin (1:5000 dilution) from Sigma; anti-Smad1 (Cat # 6944, 1:500 dilution), anti-Smad2 (Cat # 5339, 1:500 dilution) and anti-Smad3 (Cat # 9523, 1:500 dilution) from Cell Signaling; and anti-Ash2L (Cat # ab50699, 1:500 dilution) from Abcam.
4
Comprehensive Protein Marker Analysis
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Anti-Flag (F-3165, 1:5000, Sigma), anti-HA (SC-7392, 1:2000, Santa Cruz Biotechnology), anti-MYC (AE010, 1:1000, Abclonal Technology), Anti-TUBULIN (SC-23948, 1:10000, Santa Cruz Biotechnology). Anti-Aggrecan (13880-1-AP,1:1000, Proteintech); Anti-MMP13 (18165-1-AP, 1:1000, Abcam); Anti-ADAMTS5 (PA5-14350, 1:1000, Thermo); Anti-ZMPSTE24(A-8858, IHC 1:50, ABclonal). Anti-H3K27me1 (2500674, 1:1000, Millipore), Anti-H3K27me2 (ab24684, 1:1000, Abcam), Anti-H3K27me3 (07-449, 1:1000, Millipore), anti-EZH2 (5246, 1:1000, CST), anti-Suz12 (sc-271325, 1:1000, Santa Cruz), anti-EED (2514035, 1:1000, Millipore).
5
Chromatin extraction and immunoblotting
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The nuclei were extracted from 2-week-old seedlings with isolation buffer (0.25 M sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Triton X-100, and protease inhibitor cocktail) and resuspended with lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% SDS, 0.1% Na deoxycholate, 1% Triton X-100, and protease inhibitor cocktail) to release the total chromatin. The supernatant was boiled with SDS loading buffer and subjected to immunoblotting with anti-H3 (Abcam, #ab1791, 1:1000 dilution), anti-H3K27me1 (Millipore, #07-448, 1:1000 dilution), H3K27me2 (Millipore, #07-452, 1:1000 dilution), H3K27me3 (Millipore, #07-449, 1:1000 dilution), anti-H3K4me1 (Millipore, #07-436, 1:1000 dilution), anti-H3K4me2 (Millipore, #07-030, 1:1000 dilution), anti-H3K4me3 (Millipore, #07-473, 1:1000 dilution), and anti-H2AKub (Cell Signaling, #8240, 1:1000 dilution) antibodies.
6
Histone Modifications Western Blot Analysis
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Cells were washed with PBS and collected in Laemmli buffer directly. Protein samples were separated by 4-20% SDS-PAGE precast gel (Bio-Rad #456-1096) at 200 V for 30 min, and then transferred to nitrocellulose membranes (Whatman, Dassel, Germany) at 30V for 70 min. Membranes were blocked with 4% non-fat milk in PBS-0.1%Tween buffer (PBST) for 30min at room temperature. Primary antibodies anti-H3K27me3 (Millipore #07-449), anti-H3K27me1 (Millipore #07-448), anti-histone H3 (Abcam #ab1791), anti-JMJD3 (Abcam # ab85392), UTX (Abcam # ab36938), and anti-α-actin (Sigma #A5316) were diluted in 4% non-fat milk in PBST and incubated with membranes overnight at 4°C. Membranes were wash for 3 times with PBST and then incubated with secondary antibodies (Millipore) for 1 h at room temperature. Membranes were then washed with PBST 3 times and visualized using ECL reagent. Western blots analysis was performed using software Image J.
7
Histone Modification Analysis in Arabidopsis
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Ten-day-old seedlings were ground in liquid nitrogen and the powder was boiled for 5 min in protein sample buffer. The proteins were resolved on a 15% SDS/PAGE gel and transferred onto nitrocellulose membranes (Bio-Rad). Then the membranes probed with anti-H3K27me3 (Millipore 07-449); anti-H3K27me2 (Millipore 07-452); anti-H3K27me1 (Millipore 07-448); anti-H3K4me3 (Millipore 07-473); anti-H3K4me2 (Millipore 07-030); anti-H3K4me1 (Millipore 07-436); anti-H3K9me2 (Millipore 07-441); anti-H3K9me1 (Millipore 07-450); anti-H3K36me3 (Abcam ab9050); anti-H3K36me2 (Millipore 07-274); anti-H3K36me1 (Millipore 07-548); anti-H3 (Abcam ab1791) or anti-GFP (Roche 11814460001) in TBST (137 mM NaCl, 20 mM Tris·HCl pH 7.6, 0.1% Tween-20). After three washes with TBST, the signals were detected with Immobilon Western Chemiluminescent HRP Substrate (Millipore) for histone antibodies or Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific) for GFP antibody.
8
Immunofluorescence Analysis of Nuclei
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We performed immunofluorescence as previously described45 (link). Briefly, seedlings (2 g) were cut into pieces and homogenized in the cold (4 °C) with 2 mL nucleic extraction buffer (10 mM Tris-HCl (pH 9.5), 10 mM KCl, 250 mM sucrose, 3.97 mM spermidine, 9.86 mM spermine, 0.1% β-mercaptoethanol, and 1% Triton X-100). To fix the nuclei, 2 mL of 8% formaldehyde was added to the homogenate and the sample was incubated at 4 °C for 1 h with constant shaking. The sample was filtered sequentially through 100, 53, and 20 μm filters and then centrifuged at 4 °C. The pellet was resuspended in 300 μL of nucleic extraction buffer with 4.27% sucrose and then added on top of 300 μL of nucleic extraction buffer with 29% sucrose. After centrifugation at 4 °C for 30 min, the white pellet was resuspended in 40 μL of nucleic extraction buffer. Nuclei were placed on histological slides and then immunostained with anti-H3K27me1 (07-448; Millipore; 1:200 dilution) and then incubated with rabbit Alexa488- (A23220; Abbkine; 1:200 dilution) conjugated secondary antibodies for 2 h at 37 °C. After PBS washes, DNA was counterstained using DAPI in Prolong Gold (Invitrogen). Nuclei were observed with a Nikon A1RSi+ confocal microscope. For each sample, about 300 nuclei were analyzed.
9
Chromatin immunoprecipitation (ChIP) protocol
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The ChIP assays were performed as described (Castillo-Gonzalez et al., 2015) . Three grams of 10-day-old seedlings were crosslinked with 1% formaldehyde by vacuum infiltration at 4 C (10 min for histone modifications and 30 min for P SE -FM-SE, P ATXR5 -FM-ATXR5 transgenic plants and anti-SE ChIP), and the reaction was stopped with 100 mM Glycine. For histone modifications ChIP, Dynabeads Protein A or G Magnetic Beads (Thermo Fisher Scientific) with the anti-H3K9me2 (Abcam, cat #Ab1220), H3 (Millipore, Cat# MABE923), and anti-H3K27me1 (Millipore, cat #07-448) antibodies were used; Anti-FLAG M2 magnetic beads (Sigma-Aldrich, M8823), Anti-Myc Affinity Gel (Sigma-Aldrich, #A7470), and Dynabeads Protein A beads with anti-SE (Agrisera, AS09 532A) were used for FM-SE, FM-ATXR5 and SE ChIP, respectively. At least two biological replicates ChIP experiments were performed.
Quantitative PCR and RT-PCR Expression levels of the tested genes were examined by RT-PCR. Total RNAs were prepared from 10-day-old seedlings and treated with DNase before cDNA synthesis using Superscript III reverse transcriptase (Invitrogen) primed by random primers. The Actin gene was included as an internal control for normalization. The enrichment levels of specific loci after ChIP assays were also tested by quantitative PCR with CFX384 TouchÔ Real-Time PCR Detection System (BioRad). Primers are listed in Table S6.
Quantitative PCR and RT-PCR Expression levels of the tested genes were examined by RT-PCR. Total RNAs were prepared from 10-day-old seedlings and treated with DNase before cDNA synthesis using Superscript III reverse transcriptase (Invitrogen) primed by random primers. The Actin gene was included as an internal control for normalization. The enrichment levels of specific loci after ChIP assays were also tested by quantitative PCR with CFX384 TouchÔ Real-Time PCR Detection System (BioRad). Primers are listed in Table S6.
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