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Article . 2018 . Peer-reviewed
License: CC BY
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Chromatin Immunoprecipitation (ChIP) Assay Protocol v3

Authors: Kelsey Knight;

Chromatin Immunoprecipitation (ChIP) Assay Protocol v3

Abstract

Reagents - Basic cell culture media: respective to the cell line used and experimental design Distilled water: Invitrogen® UltraPure™ Distilled Water (catalog number 19977015) or equivalent product from other vendor. Highthroughput (HT) Pro A or G 96 well plate or Spin Column (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) 96-well collection plate: USA scientific® TempPlate non-skirted 96-well PCR plate natural (catalog number 1402-9596) or equivalent product from other vendor. QIAquick™ PCR purification kit (Qiagen®; catalog number 28104) or equivalent product from other vendor. Hypotonic Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) Digestion Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) Lysis Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) Column Conditioning Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) Wash Buffer 1 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) Wash Buffer 2 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) Wash Buffer 3 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) Elution Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163) 5M NaCl: dissolve 292g of NaCl in 800mL of H2O. Adjust the volume to 1L with H2O. 1M NaHCO3: dissolve 12.6g of NaHCO3 in 100 mL of H2O. Adjust the volume to 150 mL with H2O. Protease Inhibitor Cocktail (PIC): Cell signaling Technology® (catalog number 5871) or equivalent product from other vendor. Proteinase K: Qiagen® (catalog number 19133) or equivalent product from other vendor. Proteinase K stop solution: 100mM Phenylmethylsulfonyl fluoride (PMSF). Weigh 1.742g PMSF (Sigma-Aldrich®, catalog number 10837091001) and add DMSO (Sigma-Aldrich®, catalog number D2650) to a final volume of 100mL. Dissolve completely. PBS: HyClone™ Phosphate Buffered Saline (1X) (catalog number SH30256.01) or equivalent product from other vendor. Formaldehyde: VWR® 37% w/w aqueous solution (catalog number 97064-604) or equivalent product from other vendor. 0.65M Glycine: Dissolve 488g of glycine into 1L of H2O to make 6.5M glycine (10X). Dilute with PBS to make 1X before each experiment. Micrococcal Nuclease: Cell Signaling Technologies® (catalog number 10011S) or equivalent product from other vendor. 1.3-1.5% Agarose gel: dissolve 1.3-1.5g of agarose powder into 100mL of TAE buffer by microwave heating. Visualize DNA under UV light by adding appropriate amount of Ethidium Bromide, or use an equivalent method. Add appropriate amount of Ethidium Bromide to cooled down gel solution to visualize DNA under UV light. 0.5M EDTA: Dissolve 186.1g EDTA into 700mL of H2O. Adjust the volume to 1L with H2O. 100bp ladder: NEB® (catalog number N0467S) or equivalent product from other vendor. Materials - Heat block Rotator Centrifuge Microcentrifuge 37ºC incubator Nanodrop Sonicator UV light imager Rocking platform

  • BIP!
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    popularity
    This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
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    influence
    This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
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    impulse
    This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
    Average
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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
0
Average
Average
Average
hybrid