For ChIP-seq, ∼10 × 106 of untreated or stimulated (1 or 4 h after LPS) RAW cells were used for each experiment. Cells were crosslinked with 0.75% formaldehyde for 10 min at room temperature, and chromatin was sonicated as described previously (29 (link)). Each chromatin input was immunoprecipitated with 10 μg of the following antibodies: H3K4me1 (Abcam 8895), H3K4me3 (Active Motif 39159), H3K36me2 (Abcam 9049), H3K79me2 (Abcam ab3594), H3K36me3 (Abcam 9050), H3K27ac (Abcam 4729), H3K27me3 (Cell Signalling 9733) and Pu.1 (Santa Cruz sc-352). After immunoprecipitation, beads were washed three times in buffer A (20 mM Tris–HCl [pH 7.6], 2 mM EDTA, 0.1% SDS, 1% Triton-100 and 150 mM NaCl), once in buffer B (20 mM Tris–HCl [pH 7.6], 2 mM EDTA, 0.1% SDS, 1% Triton-100 and 300 mM NaCl) and once in TE containing 50 mM NaCl. DNA was eluted in TE containing 2% SDS and de-crosslinked overnight at 65°C. DNA was purified by QIAquick columns (QIAGEN) and quantified with Picogreen. Sequencing libraries were generated as previously described (53 (link),54 (link)) and sequenced on an Illumina HiSeq2000. Total RNA was extracted from 2 × 106 cells using RNeasy Kit (QIAGEN) with DNase I treatment. Libraries were then prepared using TruSeq RNA sample preparation Kit (Illumina) after depleting ribosomal RNA and sequenced on an Illumina HiSeq2000.
H3k36me2
Manufactured by Abcam
Sourced in United States
H3K36me2 is a histone modification that marks the chromatin for transcriptional elongation. It is involved in maintaining the integrity of actively transcribed regions of the genome.
Automatically generated - may contain errors
Lab products found in correlation
H3k36me3, by Abcam (10 mentions)
H3k4me3, by Merck Group (6 mentions)
H3k9me3, by Merck Group (3 mentions)
H3k79me2, by Abcam (2 mentions)
H3k27me3, by Cell Signaling Technology (2 mentions)
H3k27me3, by Merck Group (2 mentions)
H3k27me3, by Abcam (2 mentions)
Whsc1l1 antibody, by Proteintech (2 mentions)
Total h3, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
H3k9ac, by Merck Group (2 mentions)
H3k9me2, by Abcam (2 mentions)
H3k9me3, by Abcam (2 mentions)
H3k4me2, by Merck Group (2 mentions)
H3k4me1, by Abcam (1 mentions)
H3k4me3, by Active Motif (1 mentions)
Qiaquick column, by Qiagen (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
14 protocols using h3k36me2
1
ChIP-seq and RNA-seq of RAW Cells
2
Histone Methylation Analysis in Zebrafish Embryos
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Approximately 30 embryos at 48 hpf (hour post fertilization) were lysed in RIPA (Beyotime Institute of Biotechnology, China) in the presence of cocktail inibitor (Invitrogen). The lysate was sonicated for 10 rounds of 3 seconds at 50% power, then the samples were mixed with 5× Laemmli sample buffer and boiled for 5 minutes. WB was carried out according to standard procedure using following antibodies: H3K36Me2 (Abcam), H3K36Me3 (Abcam), H3 (Protech) and β-actin (Protech).
3
ChIP-seq and qPCR Analysis of Chromatin Modifications
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ChIP was performed as previously described (Jamieson et al., 2016 (link)). qPCR was performed using the Quanta Biosciences PerfeCTa Sybr Green FastMix and an Applied Biosystems Step One Plus Real-Time PCR System. ChIP-libraries were prepared as previously described (Jamieson et al., 2016 (link)) and sequencing was performed using an Illumina NextSeq 500 or HiSeq 4000 sequencer with 75- or 100-nt single-end reads, respectively. All sequencing reads were mapped to the corrected N. crassa OR74A (NC12 genome) (Galazka et al., 2016 (link)) using Bowtie2 (Langmead and Salzberg, 2012 (link)). ChIP-seq read coverage was averaged, normalized, and analyzed using tools available from deepTools2 (Ramírez et al., 2016 (link)) and SAMtools (Li et al., 2009 (link)) on the open-source platform Galaxy (Afgan et al., 2016 (link)). Sequencing tracks are displayed as 25-nt-window TDF or bigWig files with the Integrative Genomics Viewer (IGV) (Robinson et al., 2011 (link)). The following antibodies were used for ChIP: H3K27me3 (Millipore, Cat#07 – 449), H3K36me3 (Abcam, Cat#ab9050), H3K36me2 (Abcam, Cat#ab9049), H3K27ac (ActiveMotif, Cat#39133), H3K27me2/3 (ActiveMotif, Cat#39535).
4
Chromatin Immunoprecipitation of FLAG-HRP2
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293T cells were transfected with FLAG-tagged HRP2 or mutated HRP2 (W21A). 24 hours later, nuclei were prepared and resuspended in 10 mM Tris–HCl pH 7.5, containing 10 mM NaCl, 3 mM MgCl2, 3 mM CaCl2, 0.1 mM PMSF and chromatin was released by digestion with micrococcal nuclease (Sigma) at 37°C for 10 min. Digestion was optimized to produce primarily mono-nucleosomes. Digestion was stopped by addition of 20 mM EDTA. Nuclear debris was removed by centrifugation and the soluble chromatin was incubated with anti-FLAG M2 affinity gel at 4°C overnight. After washing four times, SDS loading buffer was added and boiled for 10 min. The supernatant were subjected to immunoblotting. Antibodies used were FLAG-HRP (Sigma, F7425), Histone H3 (Abcam, ab1791), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), H3K4me3 (Millipore, 05-745), H3K9me3 (Millipore, 07-442) and H3K27me3 (Abcam, ab6002).
5
Antibody Validation for ChIP-seq and Western Blot
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The antibodies used in this study are as follows: for ChIP-seq, RPB1 NTD (Cell Signaling Technology, 14958, lot #1), RPB1 Ser2 (Abcam, 5095, lot #GR3225147-1), H3K36me3 (Abcam, 9050, lot #GR3257952-1), H3K36me2 (Abcam, 9049, lot #GR3236147-1), CSTF64 (Bethyl Laboratories, A301-92A, lot #A301-092A-2), and CPSF73 (Bethyl Laboratories, A301-019A, lot #A301-091A-1); for Western blot, INTS11 (Atlas Antibodies, HPA029025, lot #A107128), lamin A (Active Motif, 39961, lot #33310001), TFIIB (Cell Signaling Technology, 4169s, lot #1), CPSF100 (Bethyl Laboratories, A301-583A-M, lot #A301-583A-M-1), CSTF50 (Bethyl Laboratories, A301-250A-M, lot #A301-250A-M-3), CSTF64 (Bethyl Laboratories, A301-092A-M, lot #A301-092A-M-2), CPSF73 (Bethyl Laboratories, A301-091A-M, lot #A301-091A-M-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, ab8245, lot #GR3317834-1).
6
Immunoblot Analysis of Histone Modifications
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Immunoblotting was performed as previously described (Honda and Selker, 2008 (link)). Briefly, Neurospora extracts were produced by sonication in extraction buffer (50 mM Hepes pH7.5, 1 mM EDTA, 150 mM NaCl, 10% Glycerol, 0.02% NP40) supplemented with cOmplete ULTRA protease inhibitor cocktail tablets (Roche, 05892970001). The following antibodies were used for immunoblotting: H3K36me3 (Cell Signaling, Cat#4909S, Clone (D5A7)), H3K36me2 (Abcam, Cat#ab9049), Histone H3 (Abcam, Cat#ab1791), IRDye 680RD Goat-anti-Rabbit secondary antibody (Licor, Cat#926 – 68071).
7
Chromatin Immunoprecipitation Antibody Protocols
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The WHSC1L1 antibody was purchased from ProteinTech Group Inc. (Cat. No. 11345‐1‐AP). The estrogen receptor alpha antibody was purchase from Bethyl Labs (Cat. No. A300‐498A). ChIP‐grade anti‐estrogen receptor alpha antibodies were purchased from Santa Cruz (sc‐543x) and Thermo Scientific (MA5‐13065). ChIP‐grade histone antibodies were used for both ChIP and immunoblotting, and were purchased from Abcam and Millipore: total H3 (Abcam ab1791), H3K36me3 (Abcam ab9050), H3K36me2 (Abcam ab9049), and H3K4me3 (Millipore 17‐678).
8
Cross-linking and ChIP-seq protocol
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ChIP experiments were performed following the procedures described previously (36 (link)). For TY1, Hrp2 and Dpf3a ChIP, cells were double crosslinked by incubation with DMA (Sangon Biotech, Shanghai, China) for 1 h followed by treatment with 1% formaldehyde for 10 min at room temperature. For H3K36me2 and Brg1 ChIP, cells were crosslinked with 1% formaldehyde. The crosslink was quenched by adding 125 mM glycine at RT for 5 min. Cells were washed three times with PBS and lysed using ChIP lysis buffer supplemented with protease inhibitor cocktail (Roche). The chromatin were then sonicated to DNA fragment sizes of 300–500 bp with Bioruptor Sonicator. Immunoprecipitation was performed with TY1 (Sigma, SAB4800032), Hrp2 (Proteintech, 15134-1-AP), Dpf3a (Custom made), H3K36me2 (Abcam, ab9049) and Brg1 (Santa Cruz, sc-17796 X). Input was used as control for ChIP-seq. After elution and reversal cross-linking, samples were treated with RNase A at 37°C for 30 min. For ChIP-seq, libraries were constructed and sequenced on Illumina HiSeq X ten. For ChIP-qPCR, 1 μl purified DNA was used per qPCR reaction. Primers for ChIP-qPCR were listed in Supplementary Table S2 .
9
Protein Extraction and Western Blotting
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Harvested cells were incubated in RIPA buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 0.5% SDC, 1% NP-40, 1× PIC and 1 mM EDTA [pH 8.0]) with gentle agitation at 4°C for 1 h. Cell lysates were clarified by centrifugation at 18 000 g for 15 min at 4°C. After SDS-PAGE, western blotting was performed using the following antibodies: SET/TAF-Iβ (sc-133138, 1:1000), MIB1 (sc-393551, 1:1000), p21 (sc-397, 1:1000), GFP (sc-9996, 1:1000), CBX8 (sc-374332, 1:1000) and β-actin (sc-47778, 1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA); H2AK119ub (8240S, 1:5000), H3K4me3 (9751S, 1:5000) and H3K27me3 (9733S, 1:5000) from Cell Signaling Technology (Danvers, MA, USA); FLAG (F3165, 1:10 000) from Sigma-Aldrich; H3K36me1 (07-548, 1:5000), H3K36me3 (07-549, 1:5000), H3K4me2 (07-030, 1:5000), H3K9me2 (07-441, 1:5000), H3K9me3 (17–625, 1:5000), H2BK120ub (17-650, 1:5000), HA (05-904, 1:5000) and H3 (05-499, 1:5000) from Merck (Rahway, NJ, USA); RAD51 (GTX70230, 1:2500) from GeneTex (Irvine, CA, USA); H3K79me1 (ab2886, 1:5000), H3K79me2 (ab3594, 1:5000), H3K79me3 (ab2621, 1:5000) and H3K36me2 (ab9049, 1:5000) from Abcam (Cambridge, UK). β-actin (ACTIN) and histone H3 were used as loading controls.
10
Histone Modification Analysis via Western Blot
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Nuclei were isolated using the same method as ChIP. Nuclear protein was extracted by boiling nuclei with SDS loading buffer at 95 °C for 10 min. FLAG-tagged proteins were detected by a horseradish peroxidase conjugated with anti-FLAG antibody (Sigma, A8592). The following histone antibodies were used for western blot: H3K9ac (1:5,000 dilution, Millipore, 07-352), H3K4me3 (1:5,000 dilution, Millipore, 04-745), H3K9me2 (1:1,000 dilution, Abcam, ab1220), H3K36me2 (1:5,000 dilution, Abcam, ab9049), H3K36me3 (1:5,000 dilution, Abcam, ab9050), and H3 (1:7,000 dilution, Abcam, ab1791). Western blots were developed using ECL Plus Western Blotting Detection System (GE Healthcare, RPN2132). Raw images of the blots are included in Supplementary Fig 6 .
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