Micro-chromatin Immunoprecipitation (μChIP) Protocol for Real-time PCR Analysis of a Limited Amount of Cells
可对限量细胞进行qPCR分析的微染色质免疫沉淀(μChIP)方法
发布: 2016年06月20日第6卷第12期 DOI: 10.21769/BioProtoc.1846 浏览次数: 9158
评审: Salma HasanAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jul 2015
Abstract
Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is an important strategy to study gene regulation. When availability of cells is limited, however, it can be useful to focus on specific genes to investigate in depth the role of transcription factors or histone marks. Unfortunately, performing ChIP experiments to study transcription factors’ binding to DNA can be difficult when biological material is restricted. This protocol describes a robust method to perform μChIP for over-expressed or endogenous transcription factors using ~100,000 cells per ChIP experiment (Masserdotti et al., 2015). We also describe optimization steps, which we think are critical for this protocol to work and which can be used to further reduce the number of cells.
Keywords: Chromatin (染色质)
PCR (PCR)
MicroChIP (微芯片)
Limited (有限公司)
Astrocyte (星形胶质细胞)
Materials and Reagents
- Microcentrifuge tubes (siliconized polypropylene, 1.7 ml) (Sigma-Aldrich, catalog number: T3406-250EA )
- Dynabeads® Protein G (Thermo Fisher Scientific, NovexTM, catalog number: 10004D )
- SYBR® green master mix (choose according to the Real-time PCR system used)
- Protease cocktail inhibitor (Roche Diagnostics, catalog number: 04693116001 )
- 37% formaldehyde solution (Sigma-Aldrich, catalog number: F8775 )
- Di(N-succinimidyl) glutarate (Sigma-Aldrich, catalog number: 80424 )
- Glycine (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 10070150 )
Note: 20x glycine stock solution (2.5 M) in water - DNase-free RNase A (ThermoFisher Scientific, catalog number: EN0531 )
- Proteinase K (Thermo Fisher Scientific, AmbionTM, catalog number: AM2546 )
- 8 M lithium chloride solution (LiCl) (Sigma-Aldrich, catalog number: L7026 )
- DynaMag®-2 magnet (Thermo Fisher Scientific, NovexTM, catalog number: 12321D )
- DNA MinElute kit (QIAGEN, catalog number: 28004 )
- Histone mark H3K9Acetyl antibody (Abcam plc, catalog number: ab4441 )
- HA antibody (Abcam plc, catalog number: ab9110 )
- SDS (Sigma-Aldrich, catalog number: L3771 )
- EDTA (Sigma-Aldrich, catalog number: 798681 )
- Tris (TRIZMA® base) (Sigma-Aldrich, catalog number: T1503 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3608 )
Note: 250 mg/ml BSA stock solution in water - HEPES (Sigma-Aldrich, catalog number: H3375 )
- NP-40 (Ipegal®) (Sigma-Aldrich, catalog number: I-3021 )
- Na-DOC (Sigma-Aldrich, catalog number: D6750 )
- NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
- ChIP IP buffer (see Recipes)
- Lysis buffer (see Recipes)
- Lithium chloride (LiCl) washing buffer (see Recipes)
- Tris-EDTA (TE) buffer (see Recipes)
- Elution buffer (see Recipes)
Equipment
- Rotating wheel
- Dry heat block
- Thermomixer (Eppendorf AG)
- Nanodrop
- Water bath sonicator (Diagenode, Bioruptor®)
- Real-time PCR instrument
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Gillotin, S. and Guillemot, F. (2016). Micro-chromatin Immunoprecipitation (μChIP) Protocol for Real-time PCR Analysis of a Limited Amount of Cells. Bio-protocol 6(12): e1846. DOI: 10.21769/BioProtoc.1846.
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分类
分子生物学 > DNA > DNA-蛋白质相互作用
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