Single-cell Visualization of Chromosome Transcriptional Territories by RNA-paint
通过RNA染色显示单细胞的染色体转录区
发布: 2016年09月05日第6卷第17期 DOI: 10.21769/BioProtoc.1914 浏览次数: 8803
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2015
Abstract
We developed a FISH-based method to directly assess chromosome-wide transcriptional activity, thereby enabling the visualization of the actively transcribed fraction of a chromosome at the single-cell level. We applied this method to probe the activity of X-chromosomes and its instability in the context of human embryonic stem cells and cancer cells.
Keywords: Transcription (转录)
Immuno-fluorescence (免疫荧光)
Chromosome-wide (染色体范围)
Materials and Reagents
- 24-well plates (Sigma-Aldrich, catalog number: Z707791-126EA )
- 13 mm round coverslips (Thermo Fisher Scientific, catalog number: 174950 )
- Glass slides
- Filter (0.2 μm)
- Filtration unit (Merck Millipore, catalog number: SCGPS05RE )
- Human embryonic stem cells (H9, WIBR2 and HUES1) and cancer cells (TCCSUP and RT112)
- Matrigel
- PBS solution (Life Technologies)
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Sucrose (Sigma-Aldrich, catalog number: S0389 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
- PIPES (Sigma-Aldrich, catalog number: P6757 )
- NaOH
- Triton X-100 (Sigma-Aldrich, catalog number: X100 )
- Ribonucleoside vanadyl complex (New England Biolabs, catalog number: S1402 )
- Paraformaldehyde (PFA) (16%, EM grade) (VWR International, catalog number: 100503-916 )
- EtOH
- Human Cot-1 DNA (Life Technologies)
- Sodium acetate anhydrous (NaOAc) (Sigma-Aldrich, catalog number: W302406 )
- Deionized formamide (Sigma-Aldrich, catalog number: F9037 )
- Rubber cement
- 20x SSC (Sigma-Aldrich, catalog number: 93017 )
- 20 mg/ml BSA (New England Biolabs, catalog number: B9000S )
- Dextran sulfate (Sigma-Aldrich, catalog number: 67578 )
- Formamide (Sigma-Aldrich, catalog number: 47670 )
- Mounting medium (Vector Laboratories, catalog number: H-1200 )
- Cy3-labelled human X-Chromosome paint (Metasystem, catalog number: D-0323-050-OR )
- FITC-labelled human X-Chromosome paint (Metasystem, catalog number: D-0323-050-FI )
- CSK buffer (see Recipes)
- 3% PFA/PBS solution (see Recipes)
- Hybridisation buffer (2x) (see Recipes)
- Washing solution (50% formamide/2x SSC) (see Recipes)
- Denaturating solution (70% formamide/2x SSC) (see Recipes)
Equipment
- Hybridisation table (Boekel Scientific, model: 240000 )
- Shake 'N BakeTM hybridisation oven (Boekel Scientific, model: 136400 )
- Centrifuge (VWR, Ependorf®, model: 5417R )
- Millivac-mini vacuum pump (Merck Millipore, catalog number: XF5423050 )
- Ependorf® Thermomixer® R (Sigma-Aldrich, catalog number: T3317 )
Note: This product has been discontinued. - Fluorescent microscope (motorized stage) (Leica, model: DMI-6000 )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Vallot, C. and Rougeulle, C. (2016). Single-cell Visualization of Chromosome Transcriptional Territories by RNA-paint. Bio-protocol 6(17): e1914. DOI: 10.21769/BioProtoc.1914.
Download Citation in RIS Format
分类
干细胞 > 胚胎干细胞 > 基于细胞的分析方法
分子生物学 > DNA > 基因表达
分子生物学 > RNA > 转录
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