10 million TC32 cells were incubated with trabectedin or DMSO for the indicated time, washed, cross-linked in 1% formaldehyde for 10 minutes and quenched with 0.2 M glycine. The cells were collected in cold PBS with 1× protease inhibitor (Sigma Aldrich), lysed in 20 mM Tri-HCl (pH 7.5), 85 mM KCl, and 0.5% NP-40 for 15 minutes on ice with dounce homogenizing. Chromatin was sheared with the E220 evolution focused sonicator (Covaris) for 10 minutes. 10 μg solubilized chromatin was immunoprecipitated with 1 μg mouse IgG (Abcam #18394), or H3K27me3 (Abcam #6002), 1 μg rabbit IgG (Cell Signaling #2729S) or 1 μg H3K9me3 (Abcam #8898), 2 μg rabbit IgG or 1 μg SMARCC1/BAF155 (Cell Signaling #11956S). Antibody-chromatin complexes were immunoprecipitated with Magna ChIP Protein A+G magnetic beads (EMD Millipore) and washed. DNA was eluted with 100mM NaHCO3, 1% SDS, and 1× proteinase K for 2-hours at 65°C followed by 10-minute incubation at 95°C. ChIP DNA was purified with QiaQuick purification kit (Qiagen). Purified SMARCC1 ChIP DNA was analyzed with ChIP-qPCR, described below. Purified H3K27me3 and H3K9me3 ChIP DNA was submitted for 2×75bp sequencing and analyzed as described below.
Magna chip protein a g magnetic beads
Manufactured by Merck Group
Sourced in United States
Magna ChIP Protein A/G Magnetic Beads are a laboratory product manufactured by Merck Group. They are magnetic beads coated with Protein A/G, which is a recombinant fusion protein that binds to the Fc region of antibodies. These beads are designed for use in chromatin immunoprecipitation (ChIP) applications.
Automatically generated - may contain errors
Lab products found in correlation
Pcr purification kit, by Qiagen (3 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (3 mentions)
Dna free dnase treatment removal 1 kit, by Thermo Fisher Scientific (3 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Chip grade tead4 antibody, by Abcam (2 mentions)
Qiaquick purification kit, by Qiagen (2 mentions)
Anti m6a antibody, by Merck Group (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Sc 2027, by Santa Cruz Biotechnology (2 mentions)
Ez nuclear rip cross linked kit, by Merck Group (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Smarcc1 baf155, by Cell Signaling Technology (1 mentions)
H3k27me3, by Abcam (1 mentions)
H3k9me3, by Abcam (1 mentions)
Rna fragmentation reagent, by Thermo Fisher Scientific (1 mentions)
Lunascript rt supermix kit, by New England Biolabs (1 mentions)
Mouse igg control antibody, by Santa Cruz Biotechnology (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
37 protocols using magna chip protein a g magnetic beads
1
Chromatin Immunoprecipitation and Sequencing
2
m6A RNA Immunoprecipitation and Quantification
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m6A IPs were carried out as described previously [32 (link)]. Briefly, TREx-BCBL1-Rta cell total RNA was fragmented into 100–200 nucleotide segments using RNA fragmentation reagents (Ambion, Austin, TX, USA) and sodium-acetate-precipitated overnight at −80 °C. A 5% input was collected prior to immunoprecipitation. The remaining RNA was combined with 25 μL of Magna ChIP Protein A+G magnetic beads (Merck Millipore, Burlington, MA, USA) coated in 5 μL of anti-m6A antibody (Merck Millipore, Burlington, MA, USA) and incubated at 4 °C overnight with rotation. RNA from inputs and m6A immunoprecipitations was eluted using proteinase K (ThermoFisher, Waltham, MA, USA) followed by Trizol LS: chloroform extraction. RNA immunoprecipitations and inputs were converted to cDNA using the LunaScript RT SuperMix kit (NEB, Hitchin, UK). m6A-immunoprecipitated samples were normalised to their respective input samples, and m6A content at a particular region was calculated relative to an unmodified control region within the same transcript.
3
TEAD4 Chromatin Immunoprecipitation Assay
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Cells were crosslinked in 0.75% formaldehyde for 15 min, then glycine was added to a final concentration of 125 mM for 5 min. After wash with cold PBS, cells were collected in PBS and sonicated on an ultrasonic homogenizer for 10 min at 20% power on ice to shear DNA to an average fragment size of 200–1000 bp. Fifty μL of each sonicated sample was removed to determine DNA concentration and fragment size. Cell lysates were incubated overnight with 20 μL Magna ChIP™ Protein A+G Magnetic Beads (EMD Millipore, Burlington, MA, USA) and 10 µg ChIP grade TEAD4 antibody (Abcam) at 4°C. Beads were collected, washed and treated with Proteinase K for 2 h at 60°C and RNase for 1 h at 37°C. DNA was purified with a PCR purification kit (Qiagen, Germantown, MD, USA). DNA fragments were assessed by qRT–PCR using the primer sequences listed in Supplementary Table 2 . Samples were normalized to input DNA.
4
TEAD4 Chromatin Immunoprecipitation Assay
5
ChIP Assay Protocol with Modifications
6
ChIP-seq of CmC3H1 transcription factor in chrysanthemum
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Chromatin immunoprecipitation experiments were carried out following a previously described protocol (Saleh et al., 2008 (link)). The full‐length sequence of CmC3H1 without the stop codon was inserted into the pSuper1300 (GFP‐C) vector using XbaI and KpnI. The resulting constructs and empty vector control were separately introduced into A. tumefaciens strain EHA105. Afterward, Agrobacterium cultures were harvested by centrifugation, resuspended in infiltration buffer (10 mm MES, 10 mm MgCl2, and 200 mm AS, pH 5.6) to a final OD600 of 1.0, and infiltrated into chrysanthemum leaves using a needleless syringe. After 3 days, approximately 1.5 g of young leaves was fixed by incubation in 1% formaldehyde under vacuum for 10 min. The reaction was stopped by adding 2.5 ml 2 m glycine (0.125 mm final concentration) for another 5 min under vacuum. The leaves were washed twice with deionized water and frozen in liquid nitrogen. Chromatin was then extracted and sonicated, followed by overnight immunoprecipitation using anti‐GFP (BE2001, Easybio, Beijing, China) and Magna ChIP™ Protein A + G Magnetic Beads (EMD Millipore, USA). The co‐precipitated DNA was purified with a QIAquick PCR Purification Kit (Qiagen GmbH, Germany). qRT‐PCR was conducted to measure the enrichment of DNA fragments. Primers are listed in Table S1 .
7
CD8+ T Cell Chromatin Immunoprecipitation
8
ChIP-seq Protocol for Histone Modifications
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ChIP experiments were performed in HEK293 cells according to the X-ChIP abcam protocol. Briefly, ∼25 μg of sheared DNA was used per IP and incubated overnight with 3 μg of H3K27me3 (PA5_31817, Thermo Fisher Scientific), 2 μg of H3K27ac (Ab4729, Abcam) or 2 μg of H3K4me3 (Ab213224, Abcam) antibodies/20 μl of Magna ChIP™ Protein A+G Magnetic Beads (Merck Millipore) complexes. The following day, the beads were subsequently washed in low salt wash (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0, 500 mM NaCl) and LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8.0). Samples were then treated with 200 μg of proteinase K in a total volume of 200 μl for 45 min at 50°C. DNA was prepared using the GeneJET Gel Extraction kit (Thermo Fisher Scientific) according to the manufacturer's recommendations, eluted in 15 μl of elution buffer and diluted two times with water. Primer list used can be found in Supplementary Table S6 .
9
RARγ Cistrome Analysis in BAC-RARG-EGFP RWPE-1 Cells
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ChIP was performed in BAC-RARG-EGFP containing RWPE-1 cells in the presence of CD437 (10 nM, 2 h) or DMSO as previously described [6 (link)]. Briefly, triplicate cultures of approximately 20 × 106 cells were crosslinked with 1% formaldehyde solution, quenched with glycine (0.125 M) and harvested in cold PBS. Sonication of crosslinked chromatin was performed using a Bioruptor® UCD-200 TM Sonicator (Diagenode) for a total of 15 min. Immunoprecipitation of sonicated material was performed with antibodies against enhanced green fluorescent protein (ab290, Abcam) or IgG (sc-2027×, Santa Cruz) for 16 h, and antibody/bead complexes isolated with Magna ChIPTM Protein A+G magnetic beads (Millipore). Complexes were washed, reverse crosslinked and treated sequentially with RNase and proteinase K prior to DNA isolation. Sequencing (100 bp single end, >30 × 106 average reads/sample) was performed at the RPCCC Genomics Shared Resource core facility. The RARγ cistrome was analyzed with Rsubread/csaw [89 ], along with TF motif analyses (MotifDb). Binding site overlaps between RARγ and the other cistromes were tested (ChIPpeakAnno [90 (link)] and bedtools). Peak density plots were performed using the annotatePeaks.pl tool available from the HOMER (Hypergeometric Optimization of Motif EnRichment) suite.
10
ChIP-qPCR Analysis of FOXO3a Binding in Bone Marrow
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ChIP was carried out in WT total bone marrow TER119+ cells as previously described [17 (link)]. Briefly, cells were cross-linked in 0.4% formaldehyde in PBS, and lysed (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% NP40). Lysate was sonicated for 30 cycles of 30 s on/30 s off at 4C° using a Bioruptor Standard sonication device (Diagenode). The cell lysate was then diluted in ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA 150 mM NaCl, 1% Triton, 0.01% SDS) and incubated at 4C° overnight with anti-FOXO3a antibody (Millipore #07–702) and Magna ChIPTM Protein A+G magnetic beads (Millipore #16–663). Beads were then washed (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% Triton, 0.1% SDS) and recovered. The antibody-protein-DNA complexes were reverse cross-linked for DNA isolation and quantitative PCR (qPCR) analysis. Foxo3-/- TER119+ cells were used as negative controls. Putative binding sites were located using MatInspector from Genomatix (http://www.genomatix.de/ ). Primer specific sequences are listed in S11 Table . See S12 Table for all antibodies.
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