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Magna chip g tissue kit

Manufactured by Merck Group
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Sourced in Germany, United States

The Magna ChIP G tissue kit is a laboratory equipment product designed for chromatin immunoprecipitation (ChIP) assays. It is intended to be used for the isolation and purification of chromatin from tissue samples.

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Lab products found in correlation

Water bath sonicator, by Qsonica (2 mentions) Ab124055, by Abcam (2 mentions) Total h3, by Cell Signaling Technology (2 mentions) H3k9ac, by Active Motif (2 mentions) H3k27me3, by Cell Signaling Technology (2 mentions) H3k4me3, by Cell Signaling Technology (2 mentions) H3k9me2, by Abcam (2 mentions) Nuclear lysis buffer, by Merck Group (2 mentions) Sybr green reagent, by Qiagen (2 mentions) Bioruptor plus, by Diagenode (2 mentions) Rabbit igg, by Merck Group (2 mentions) Epitect chip qpcr primer, by Qiagen (1 mentions) Anti p65, by Abcam (1 mentions) Anti p50, by Cell Signaling Technology (1 mentions) Dr gentle precipitation carrier kit, by Takara Bio (1 mentions) Novaseq 6000 sequencer, by Illumina (1 mentions) Anti p50, by Abcam (1 mentions)

10 protocols using magna chip g tissue kit

1

Chromatin Immunoprecipitation from Rat DRG Tissue

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We performed chromatin immunoprecipitation (ChIP) using the Magna ChIP G tissue kit (Millipore; catalog #17-20000), according to the manufacturer’s instructions. Briefly, fresh DRG tissues were rapidly removed from anesthetized rats and were stabilized for 3 min using stabilization buffer. Then, the DRGs were incubated in 2% formaldehyde for 20 min at ~26 °C. After being washed three times with PBS, the DRGs were incubated in lysis buffer for 15 min on ice. Finally, the DRG tissues were sonicated (30 s on and 30 s off, repeated 200 times) in ChIP dilution buffer using a water bath sonicator (Qsonica, Newtown, CT) at 4 °C. The sonicated DRG samples were centrifuged at 12,000 rpm for 10 min at 4 °C. Chromatin was pulled down using the following antibodies: IgG (as a negative control; catalog #ab124055, Abcam), total H3 (catalog #2650s, Cell Signaling Technology), H3K9me2 (catalog #ab1220, Abcam), H3K9ac (catalog #39917, Active Motif), H3K27me3 (catalog #9773s, Cell Signaling Technology), and H3K4me3 (catalog #9751s, Cell Signaling Technology). After chromatin precipitation, we performed quantitative PCR using the primers described in Supplementary Table 4. Data were analyzed and corrected by input and total H3 conditions 60 (link).
Laumet G., Garriga J., Chen S.R., Zhang Y., Li D.P., Smith T.M., Dong Y., Jelinek J., Cesaroni M., Issa J.P, & Pan H.L. (2015). G9a Is Essential for Epigenetic Silencing of K+ Channel Genes in Acute-to-Chronic Pain Transition. Nature neuroscience, 18(12), 1746-1755.
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2

Chromatin Immunoprecipitation of Liver Samples

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Chromatin immunoprecipitation assay was using by Magna ChIP G Tissue Kit (Millipore, 17-20000). Livers from wild-type female mice fasted overnight were collected and mildly dissociated by dounce with pestle A for six strokes in PBS containing 1% formaldehyde and rocked for 15 min, quenched with glycine, washed with PBS, and sonicated with a probe-type sonifier in tissue lysis buffer supplemented with protease inhibitors and PMSF. Sonicated extracts contained 10 μg cross-linked chromatin were immunoprecipitated with antibodies for ERα (6.7 μL), ERRα (10 μL) or IgG (2 μL, Cell signaling, 2729, lot# 8), and captured with protein G magnetic beads. DNA was purified using spin columns. Three sets of input and immunoprecipitated samples from three mice were pooled. Sample was analyzed by real time PCR and the amount of immunoprecipitated DNA in each sample was presented as fold relative enrichment to IgG-associated DNA by using the delta Ct (ΔCt) method. Primer sequences are listed in Table S5.
Yang M., Liu Q., Huang T., Tan W., Qu L., Chen T., Pan H., Chen L., Liu J., Wong C.W., Lu W.W, & Guan M. (2020). Dysfunction of estrogen-related receptor alpha-dependent hepatic VLDL secretion contributes to sex disparity in NAFLD/NASH development. Theranostics, 10(24), 10874-10891.
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3

Chromatin Immunoprecipitation Assay Protocol

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The commercially available Magna ChIP™ G Tissue Kit (17-20000, Millipore) was used, and the published protocol was followed (Montalvo-Ortiz et al. 2014 (link)). Fragmented chromatin lysate (400–600 bp size determined by agarose gel electrophoresis) was immunoprecipitated with 5μg of antibody directed against H3K27ac, H3K9ac, H4K12ac or H3K27me3. The DNA–histone complex was incubated with salmon sperm DNA/protein A-agarose beads for 1 hour to estimate non-specific binding. The DNA–histone complex was eluted from the beads and dissociated at 65°C for 4 hours under high-salt conditions. Proteins were digested using proteinase K treatment, and the associated DNA was precipitated with 100% ethanol and resuspended in 80 μl of PCR grade water.
Montalvo-Ortiz J.L., Fisher D.W., Rodriguez G., Fang D., Csernansky J.G, & Dong H. (2017). Histone Deacetylase Inhibitors Reverse Age-Related Increases in Side Effects of Haloperidol in Mice. Psychopharmacology, 234(16), 2385-2398.
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4

Chromatin Immunoprecipitation of NF-κB in Neonatal Lungs

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For each ChIP experiment one set (right and left) of neonatal lungs was used. Chromatin was prepared using the Magna ChIP G tissue kit (Millipore) per protocol with the notable modification of sonicating tissues in nuclear lysis buffer (Millipore). Sonication was performed using the Diagenode Bioruptor Plus for three sets of ten 30 s cycles. DNA was quantified using a spectrophotometer. Twenty-five microgram of chromatin was diluted to a total volume of 500 μL in dilution buffer. Antibodies used for immunoprecipitation included Rabbit IgG (Millipore), anti-p65 (Abcam, Ab7970), and anti-p50 (Cell Signaling, D4P4D). Immunoprecipitations were incubated at 4°C overnight. Enrichment of the IL-1α promoter was assessed by real-time qPCR using SYBR green reagent (Qiagen) and a predesigned IL-1α promoter spanning primer (Qiagen EpiTect ChIP qPCR primer, NM_ 010554.4 (-1)01Kb). Results were expressed as percent input.
Butler B., De Dios R., Nguyen L., McKenna S., Ghosh S, & Wright C.J. (2019). Developmentally Regulated Innate Immune NFκB Signaling Mediates IL-1α Expression in the Perinatal Murine Lung. Frontiers in Immunology, 10, 1555.
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5

ChIP Assay for Omi/HtrA2 Promoter

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The ChIP assay was performed according to the instructions of the Magna ChIP G Tissue Kit (#17-20000; millipore EZ-ChIP, Darmstadt, Germany). Briefly, mouse heart were treated with 1% (w/v) formaldehyde for 15 min. Cross-linked chromatin was then prepared and sonicated to an average size of 500 bp. DNA fragments were immunoprecipitated with antibodies specific to HSF1 (#4356; Cell Signaling Technology, Danvers, MA, USA), p53 (#ab28; Abcam, Cambridge, UK), or control rabbit, mouse IgG at 4 °C overnight. After reversal of cross-linking, the immunoprecipitated chromatin was amplified by primers corresponding to core regions of the Omi/HtrA2 promoter. Primers were designed and synthesized as follow: Omi promoter-F, 5’-GCTACCGTCGTGCCCTGCTT-3’; Omi promoter-R, 5’-ATGCCCGAAGGCTCCAGTTT-3’.
Liu D., Liu X., Wu Y., Wang W., Ma X, & Liu H. (2016). Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter. International Journal of Molecular Sciences, 17(1), 119.
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6

Chromatin Immunoprecipitation Sequencing

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ChIP-seq assays were performed with Magna ChIP™ G Tissue Kit (Millipore, USA). The tissue was fixed in 1% formaldehyde for 10 min at room temperature, after which 0.125 M glycine was added and the mixture was sat for 5 min to terminate the crosslinking reaction. The tissue was then treated with cell lysis buffer and nucleus was collected by centrifuging at 800 g for 5 min at 4 °C. Next, nucleus was treated with nucleus lysis buffer and sonicated to fragment chromatin DNA. Antibodies against H3K27ac (10 μg, Abcam, USA) were incubated overnight with the crosslinked protein and DNA for immunoprecipitation reactions with protein A/G magnetic beads. DNA fragments were purified and collected by a Dr.GenTLE Precipitation Carrier kit (Takara, Japan). The purified DNA library was then sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model by SeqHealth (Wuhan, China). We filtered, aligned and processed the data to generate BAM files as described in ATAC-seq.
Ying P., Chen C., Lu Z., Chen S., Zhang M., Cai Y., Zhang F., Huang J., Fan L., Ning C., Li Y., Wang W., Geng H., Liu Y., Tian W., Yang Z., Liu J., Huang C., Yang X., Xu B., Li H., Zhu X., Li N., Li B., Wei Y., Zhu Y., Tian J, & Miao X. (2023). Genome-wide enhancer-gene regulatory maps link causal variants to target genes underlying human cancer risk. Nature Communications, 14, 5958.
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7

Chromatin Immunoprecipitation from Rat DRG Tissue

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We performed chromatin immunoprecipitation (ChIP) using the Magna ChIP G tissue kit (Millipore; catalog #17-20000), according to the manufacturer’s instructions. Briefly, fresh DRG tissues were rapidly removed from anesthetized rats and were stabilized for 3 min using stabilization buffer. Then, the DRGs were incubated in 2% formaldehyde for 20 min at ~26 °C. After being washed three times with PBS, the DRGs were incubated in lysis buffer for 15 min on ice. Finally, the DRG tissues were sonicated (30 s on and 30 s off, repeated 200 times) in ChIP dilution buffer using a water bath sonicator (Qsonica, Newtown, CT) at 4 °C. The sonicated DRG samples were centrifuged at 12,000 rpm for 10 min at 4 °C. Chromatin was pulled down using the following antibodies: IgG (as a negative control; catalog #ab124055, Abcam), total H3 (catalog #2650s, Cell Signaling Technology), H3K9me2 (catalog #ab1220, Abcam), H3K9ac (catalog #39917, Active Motif), H3K27me3 (catalog #9773s, Cell Signaling Technology), and H3K4me3 (catalog #9751s, Cell Signaling Technology). After chromatin precipitation, we performed quantitative PCR using the primers described in Supplementary Table 4. Data were analyzed and corrected by input and total H3 conditions 60 (link).
Laumet G., Garriga J., Chen S.R., Zhang Y., Li D.P., Smith T.M., Dong Y., Jelinek J., Cesaroni M., Issa J.P, & Pan H.L. (2015). G9a Is Essential for Epigenetic Silencing of K+ Channel Genes in Acute-to-Chronic Pain Transition. Nature neuroscience, 18(12), 1746-1755.
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8

Chromatin Immunoprecipitation of RNA Polymerase

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Anti-mouse RNA polymerase III subunit RPC39 mouse monoclonal antibody was purchased from Santa Cruz (catalog no. SC-21753). Anti-mouse RNA polymerase II mouse monoclonal antibody (Cat. 39097) was from Active Motif. For chromatin immunoprecipitation (ChIP), the Magna ChIP G Tissue kit (Millipore) was used following the manufacturer’s instructions.
Li J., Kannan M., Trivett A.L., Liao H., Wu X., Akagi K, & Symer D.E. (2014). An antisense promoter in mouse L1 retrotransposon open reading frame-1 initiates expression of diverse fusion transcripts and limits retrotransposition. Nucleic Acids Research, 42(7), 4546-4562.
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9

ChIP-qPCR protocol for NF-κB p50

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Chromatin was prepared using the Magna ChIP G tissue kit (Millipore) per protocol with the notable modification of sonicating tissues in nuclear lysis buffer (Millipore). Sonication was performed using the Diagenode Bioruptor Plus for three sets of ten 30 second cycles. Proper sonication of chromatin (200–900 base pairs) was verified for reverse cross-linked DNA via electrophoresis in a 1.2% agarose gel. Chromatin was quantified using a spectrophotometer. 25 μg of chromatin was diluted to a total volume of 500 μL in dilution buffer. Antibodies used for immunoprecipitation included Rabbit IgG (Millipore), and anti-p50 (Abcam, 32360). Immunoprecipitations were incubated at 4°C overnight. Reverse cross-linking of immunoprecipitated chromatin was accomplished with a two-hour incubation at 62°C. Immunoprecipitated and purified DNA quality was assessed using a spectrophotometer. Enrichment of the CRP promoter was assessed by real-time qPCR using SYBR green reagent (Qiagen) and primers designed to span a section of the promoter region located 150 base pairs upstream and downstream of the p50 consensus sequence. Results were expressed as percent input.
Kawabata K., Ujikawa M., Egawa T., Kawamoto H., Tachibana K., Iizasa H., Katsura Y., Kishimoto T, & Nagasawa T. (1999). A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution. Proceedings of the National Academy of Sciences of the United States of America, 96(10), 5663-5667.
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10

ChIP Assay for Histone H3K27ac

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For measuring histone acetylation marks, the Magna ChIP G Tissue Kit (Millipore, 17-20000) assay was used and performed according to the kit protocol. Fragmented chromatin lysate was precipitated with 2–3 ul of antibody for H3K27ac (Abcam, ab4729). The DNA-histone complex was incubated with salmon sperm DNA/protein A-agarose beads overnight to estimate non-specific binding. The DNA-histone complex was eluted from the beads and dissociated at 65° for 4 h in a water bath under high-salt conditions. Proteins were digested using proteinase-K treatment, and the associated DNA was precipitated with 100% ethanol and re-suspended in 50 ul of PCR-grade water (14 (link), 16 (link)).
Rodriguez G., Fisher D.W., McClarty B., Montalvo-Ortiz J., Cui Q., Chan C.S, & Dong H. (2023). Histone deacetylase inhibitors mitigate antipsychotic risperidone-induced motor side effects in aged mice and in a mouse model of Alzheimer’s disease. Frontiers in Psychiatry, 13, 1020831.
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