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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   

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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

  1. Microcentrifuge tubes (siliconized polypropylene, 1.7 ml) (Sigma-Aldrich, catalog number: T3406-250EA )
  2. Dynabeads® Protein G (Thermo Fisher Scientific, NovexTM, catalog number: 10004D )
  3. SYBR® green master mix (choose according to the Real-time PCR system used)
  4. Protease cocktail inhibitor (Roche Diagnostics, catalog number: 04693116001 )
  5. 37% formaldehyde solution (Sigma-Aldrich, catalog number: F8775 )
  6. Di(N-succinimidyl) glutarate (Sigma-Aldrich, catalog number: 80424 )
  7. Glycine (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 10070150 )
    Note: 20x glycine stock solution (2.5 M) in water
  8. DNase-free RNase A (ThermoFisher Scientific, catalog number: EN0531 )
  9. Proteinase K (Thermo Fisher Scientific, AmbionTM, catalog number: AM2546 )
  10. 8 M lithium chloride solution (LiCl) (Sigma-Aldrich, catalog number: L7026 )
  11. DynaMag®-2 magnet (Thermo Fisher Scientific, NovexTM, catalog number: 12321D )
  12. DNA MinElute kit (QIAGEN, catalog number: 28004 )
  13. Histone mark H3K9Acetyl antibody (Abcam plc, catalog number: ab4441 )
  14. HA antibody (Abcam plc, catalog number: ab9110 )
  15. SDS (Sigma-Aldrich, catalog number: L3771 )
  16. EDTA (Sigma-Aldrich, catalog number: 798681 )
  17. Tris (TRIZMA® base) (Sigma-Aldrich, catalog number: T1503 )
  18. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3608 )
    Note: 250 mg/ml BSA stock solution in water
  19. HEPES (Sigma-Aldrich, catalog number: H3375 )
  20. NP-40 (Ipegal®) (Sigma-Aldrich, catalog number: I-3021 )
  21. Na-DOC (Sigma-Aldrich, catalog number: D6750 )
  22. NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
  23. ChIP IP buffer (see Recipes)
  24. Lysis buffer (see Recipes)
  25. Lithium chloride (LiCl) washing buffer (see Recipes)
  26. Tris-EDTA (TE) buffer (see Recipes)
  27. Elution buffer (see Recipes)

Equipment

  1. Rotating wheel
  2. Dry heat block
  3. Thermomixer (Eppendorf AG)
  4. Nanodrop
  5. Water bath sonicator (Diagenode, Bioruptor®)
  6. Real-time PCR instrument

Procedure

  1. Optimization steps
    These 4 steps are critical and preparatory work is required for this protocol to work optimally.
    1. The chromatin concentration per 100,000 cells has to be estimated for each cell line or tissue used. Follow the protocol up to step C11 and continue from step C28 to reverse the cross-linking, treat with RNase A and proteinase K before purifying the sample through a column. Measure the concentration of the DNA with a nanodrop.
      Note: Typical concentration is between 4-6 ng/µl with NS-5 cells giving a yield of chromatin solution between 400-600 ng per 100 μl. This information will be used to optimize the antibody concentration.
    2. The concentration of antibody to be used per 100,000 cells needs to be optimized by initially testing at least 3 different antibody concentrations. If the antibody is commercial, this can be done by working out the optimal concentration per μg of chromatin given on the manufacturer’s datasheet and the estimated chromatin concentration from the previous step. Otherwise, the antibody concentration needs to be tested empirically until an efficient immunoprecipitation is achieved.
      We have noticed a maximum antibody concentration for which the immunoprecipitation peaks (see Figures 1-2).
    3. Sonication time needs to be optimized depending on the machine used. As a guideline, in an ice-cold water bath sonicator, we sonicate a volume of 400-500 μl for maximum 10 min (30 sec on/30 sec off on maximum power) keeping a constant layer of ice. Optimal sonicated chromatin is depicted in Figure 3.
    4. Design primers with Primer3 (http://primer3.ut.ee) and test their specificity by serial dilution of genomic DNA. The amplicon size should be between 100 and 150 bp.

  2. Before starting
    1. Plate or sort 100,000 cells by flow cytometry per µChIP including mock ChIP with additional cells for input. 
      Note: Input can be made with 100,000 cells (100%) or with a fraction of cells bearing in mind the percentage for final quantification.
    2. Resuspend the Dynabeads® and take a sufficient volume for the pre-clearing step and the IP step. Capture on magnet, aspire supernatant and wash with 1 ml ice-cold ChIP IP buffer on a rotating wheel for 3 min at room temperature. Repeat 2 times and resuspend in ChIP IP buffer with the same volume initially pipetted out from stock. Put the tube on a rotating wheel at 4 °C while proceeding with the next steps.

  3. Experimental procedure
    For plated cells, take off media and wash cells with 1x PBS at room temperature. Cross-link the cells directly on plate with sufficient volume of cross-linking solution to cover cells. For sorted cells, use siliconized tubes during sorting, pellet the cells (100 x g for 3 min), wash with 500 ml PBS and cross-link in 1 ml PBS on a rotating wheel (12 rpm).
    1. (Optional) If double cross-linking is required, cross-link first with 2 mM Di(N-succinimidyl) glutarate in PBS for 45 min at room temperature with gentle rocking, then wash twice with PBS.
    2. Cross-link with 1% formaldehyde in PBS for 10 min with gentle rocking.
    3. Quench fixation with 125 mM glycine and keep on rocker for 5-10 min.
    4. Wash twice with cold PBS, on the last wash remove as much PBS as possible with a pipette.
    5. Lyse cells directly on plate with 100 µl lysis buffer per 100,000 cells (i.e., minimum 300 µl to get one input, one mock and one ChIP sample; 400 µl if 2 different proteins are immunoprecipitated).
    6. Collect cells with a cell scraper and transfer into a siliconized tube.
      Optional: Snap freeze in liquid nitrogen (this step is not required for some antibodies, such as antibodies for histone marks).
    7. Leave to thaw on ice.
    8. Sonicate in a water bath sonicator with ice for 10 min maximum (see step A3).
    9. Centrifuge at 4 °C at 16,000 x g for 5 min and transfer supernatant into a new siliconized tube.
    10. Take an aliquot (~10 µl) and run it on a 1% agarose gel to check shearing (see Figure 3 for optimal result).
      Note: Make sure the chromatin runs between 500-1,000 bp before continuing or sonicate longer.
    11. Pre-clear with 10 µl washed Dynabeads® per 100 µl of chromatin solution for 1 h on rotating wheel at 4 °C.
    12. Capture beads on magnet and collect supernatant into a fresh siliconized tube.
    13. Aliquot 100 µl in siliconized tubes for the input, the mock ChIP and each ChIP sample(s).
    14. Add 400 µl ChIP IP buffer to the mock and ChIP sample(s) (SDS concentration has to be reduced to 0.1%).
      Note: Keep the input sample(s) at -20 °C until the next day.
    15. Add correct amount of antibody to the diluted mock and ChIP samples and rotate overnight on a wheel at 4 °C.
      Note: Keep the remaining of the washed beads on a wheel overnight to fully saturate them with the BSA contained in the buffer.
    16. Next morning, add 25 µl of saturated Dynabeads® and put back on a wheel at 4 °C for 2 h.
    17. Capture the Dynabeads® on a magnet, take out the supernatant and add 1 ml of ice-cold LiCl washing buffer. Put on a wheel at room temperature for 3-5 min and repeat 4 times.
    18. Wash 1 time with 1 ml ice-cold TE buffer. Centrifuge briefly before putting on a magnet to remove as much as possible of the TE buffer.
    19. Elute antibody/chromatin complexes by adding 50 µl of elution buffer directly to washed Dynabeads®.
    20. Vortex briefly.
    21. Shake on Thermomixer at 1,000 rpm for at least 15 min at room temperature.
    22. Centrifuge at maximum speed in a top table centrifuge for 3 min.
    23. Put tubes on magnet and collect supernatants into fresh siliconized tubes.
    24. Repeat the elution step and combine elutes in the same tubes.
    25. Thaw input sample and follow next steps for all samples.
    26. Add 10 µl 5 M NaCl (final concentration 0.45 M).
    27. Boil samples in dry heat block for 15 min to reverse crosslinking.
    28. Spin down tubes at maximum speed for 30 sec at room temperature.
    29. Leave the tubes to cool down before adding 1 µl DNase-free RNase A.
    30. Incubate for 15 min at 37 °C.
    31. Add 0.5 µl proteinase K.
    32. Incubate for 15 min at 67 °C.
    33. Purify DNA with a kit designed for low DNA quantity (e.g., DNA MinElute kit).
    34. Quantities of immunoprecipitated DNA are calculated by comparison with a standard curve generated by serial dilutions of input DNA using a Real-Time PCR System and an SYBR green-based kit for quantitative PCR (Frank et al., 2001). As a guideline, we use 1-2 μl of purified DNA per reaction depending on the targeted protein and the number of genes to analyze. The final concentration of precipitated DNA varies according to the targeted protein. In NS-5 cells, we obtained around 4 ng/μl for endogenous transcription factors and up to 30 ng/μl for histone marks.

Representative data


Figure 1. μChIP of the histone mark H3K9Acetyl with increasing amount of antibody in mouse cortical astrocytes. Real-time PCR was performed in the promoter region of Pax6 (F: cggtgaaagaagccactagg, R: agggaacacaccaactttcg) and in the promoter region of T-cell receptor (TCR) (F: cttacaccccaaacctccaa, R: gggaggatgaggagaaaagg).


Figure 2. μChIP of overexpressed HA-tagged Neurogenin2 in NS-5 cells with increasing amount of antibody. Real-time PCR was performed with primers recognizing the promoter region of the Neurogenin2 (Neurog2) target gene Fbxw7 (F: gcgctttgaagaaagacctg, R: gttttcaaggggcgaatgta) and the ORF region of Dll1, not bound by Neurog2 (F: gtctcaggaccttcacagtag, R: gagcaaccttctccgtagtag).


Figure 3. Optimal chromatin solution after sonication of 500,000 astrocytes in 500 μl lysis buffer for 10 min (30 sec on/30 sec off) setting H, Diagenode Bioruptor. Two samples (10 μl each) were run on a 1% agarose gel alongside a DNA ladder.

Recipes

Note: Prepare all the solutions fresh from stock solutions before starting and keep on ice.

  1. ChIP IP buffer
    20 mM HEPES (pH 7.6) from 1 M stock solution
    0.2 M NaCl from 5 M stock solution
    2 mM EDTA (pH 8.0) from 0.5 M stock solution
    0.1% Na-DOC from 10% fresh solution
    1% Triton X-100 from 10% fresh solution
    5 mg/ml BSA from 250 mg/ml stock solution
    Supplemented with protease inhibitor cocktail
  2. Lysis buffer
    0.5% SDS from 10% stock solution
    10 mM EDTA (pH 8.0) from 0.5 M stock solution
    50 mM Tris-HCl (pH 8.0) from 1 M stock solution
    Supplemented with 1x protease inhibitor cocktail
  3. Lithium chloride (LiCl) washing buffer
    50 mM HEPES (pH 7.6) from 1 M stock solution
    1 mM EDTA (pH 8.0) from 0.5 M stock solution
    1% NP-40 from 10% fresh solution
    1% Na-DOC from 10% fresh solution
    0.5 M LiCl from 8 M stock solution
  4. Tris-EDTA (TE) buffer
    10 mM Tris-HCl (pH 8.0) from 1 M stock solution
    1 mM EDTA (pH 8.0) from 0.5 M stock solution
  5. Elution buffer
    50 mM NaHCO3 (pH 5.2) from 3 M stock solution
    1% SDS from 10% stock solution

Acknowledgments

This work was supported by a project grant from the Wellcome Trust (WT091800MA) and a Grant-in-Aid from the Medical Research Council (U117570528) to F. G. This protocol was established with methods from Peggy Farnham's laboratory and with methods published by François Guillemot's laboratory. We are grateful to Dr Koji Oishi for critical reading of the manuscript.

References

  1. Frank, S. R., Schroeder, M., Fernandez, P., Taubert, S. and Amati, B. (2001). Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. Genes Dev 15(16): 2069-2082.
  2. Masserdotti, G., Gillotin, S., Sutor, B., Drechsel, D., Irmler, M., Jorgensen, H. F., Sass, S., Theis, F. J., Beckers, J., Berninger, B., Guillemot, F. and Gotz, M. (2015). Transcriptional mechanisms of proneural factors and REST in regulating neuronal reprogramming of astrocytes. Cell Stem Cell 17(1): 74-88.

简介

染色质免疫沉淀后深层测序(ChIP-Seq)是研究基因调控的重要策略。 然而,当细胞的可用性有限时,可能有用的是专注于特定的基因深入研究转录因子或组蛋白标记的作用。 不幸的是,当生物材料受到限制时,进行ChIP实验以研究转录因子与DNA的结合可能是困难的。 该方案描述了使用约100,000个细胞/ChIP实验对过表达或内源性转录因子进行μChIP的稳健方法(Masserdotti等人,2015)。 我们还描述了优化步骤,我们认为这是协议工作的关键,可以用于进一步减少单元格的数量。

关键字:染色质, PCR, 微芯片, 有限公司, 星形胶质细胞

材料和试剂

  1. 微量离心管(硅化聚丙烯,1.7ml)(Sigma-Aldrich,目录号:T3406-250EA)
  2. Dynabeads Protein G(Thermo Fisher Scientific,Novex TM ,目录号:10004D)
  3. SYBR ®绿色主混合(根据所使用的实时PCR系统进行选择)
  4. 蛋白酶混合物抑制剂(Roche Diagnostics,目录号:04693116001)
  5. 37%甲醛溶液(Sigma-Aldrich,目录号:F8775)
  6. 二(N-琥珀酰亚胺基)戊二酸酯(Sigma-Aldrich,目录号:80424)
  7. 甘氨酸(Thermo Fisher Scientific,Fisher Scientific ,目录号:10070150)
    注意:20x甘氨酸储备液(2.5 M)的水溶液
  8. 无DNA酶的RNase A(ThermoFisher Scientific,目录号:EN0531)
  9. 蛋白酶K(Thermo Fisher Scientific,Ambion TM,目录号:AM2546)
  10. 8M氯化锂溶液(LiCl)(Sigma-Aldrich,目录号:L7026)
  11. DynaMag -2磁体(Thermo Fisher Scientific,Novex TM ,目录号:12321D)
  12. DNA MinElute试剂盒(QIAGEN,目录号:28004)
  13. 组蛋白标记H3K9乙酰基抗体(Abcam plc,目录号:ab4441)
  14. HA抗体(Abcam plc,目录号:ab9110)
  15. SDS(Sigma-Aldrich,目录号:L3771)
  16. EDTA(Sigma-Aldrich,目录号:798681)
  17. Tris(TRIZMA)碱(Sigma-Aldrich,目录号:T1503)
  18. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3608)
    注意:250 mg/ml BSA储备水溶液
  19. HEPES(Sigma-Aldrich,目录号:H3375)
  20. NP-40(Ipegal)(Sigma-Aldrich,目录号:I-3021)
  21. Na-DOC(Sigma-Aldrich,目录号:D6750)
  22. NaHCO 3(Sigma-Aldrich,目录号:S5761)
  23. ChIP IP缓冲区(请参阅配方)
  24. 裂解缓冲液(见配方)
  25. 氯化锂(LiCl)洗涤缓冲液(见配方)
  26. Tris-EDTA(TE)缓冲液(参见配方)
  27. 洗脱缓冲液(见配方)

设备

  1. 旋转轮
  2. 干热块
  3. 热固机(Eppendorf AG)
  4. Nanodrop
  5. 水浴超声器(Diagenode,Bioruptor )
  6. 实时PCR仪

程序

  1. 优化步骤
    这4个步骤是至关重​​要的,并且需要准备工作以使该协议最佳地工作。
    1. 对于每个使用的细胞系或组织,必须估计每100,000个细胞的染色质浓度。按照方案直到步骤C11并从步骤C28继续以逆转交联,在通过柱纯化样品之前用RNA酶A和蛋白酶K处理。用纳米滴测量DNA的浓度。
      注意:典型浓度为4-6ng /μl,NS-5细胞的染色质溶液产量为400-600ng /100μl。此信息将用于优化抗体浓度。
    2. 每100,000个细胞中使用的抗体的浓度需要通过最初测试至少3种不同的抗体浓度来优化。如果抗体是商业的,这可以通过制定在制造商的数据表上给出的每μg染色质的最佳浓度和来自前一步骤的估计的染色质浓度来进行。否则,抗体浓度需要根据经验进行测试,直到达到有效的免疫沉淀为止。
      我们注意到免疫沉淀峰出现的最大抗体浓度(参见图1-2)。
    3. 根据所使用的机器,需要优化超声处理时间。作为指导,在冰冷的水浴超声波仪中,我们超声400-500微升的最大10分钟(30秒开/30秒关闭最大功率)保持恒定的冰层。最佳超声处理的染色质如图3所示
    4. 使用Primer3设计引物( http://primer3.ut.ee )并测试其通过基因组DNA的连续稀释的特异性。扩增子大小应在100至150bp之间
  2. 开始之前
    1. 通过流式细胞术每μChIP平板或分选100,000个细胞,包括具有额外的细胞的模拟ChIP输入。 注意:可以输入100,000个单元格(100%),也可以输入一小部分单元格,记住最终定量的百分比。
    2. 重新悬挂Dynabeads ®,并为预清除步骤和IP步骤占用足够的体积。在磁铁上捕获上清液,并在室温下在旋转轮上用1ml冰冷的ChIP IP缓冲液洗涤3分钟。重复2次,并重悬于具有相同体积的ChIP IP缓冲液中,最初从储备液中移出。将管放在4°C的旋转轮上,同时继续下一步
  3. 实验程序
    对于平板细胞,脱离培养基并在室温下用1x PBS洗涤细胞。 用足够体积的交联溶液直接在板上交联细胞以覆盖细胞。 对于分选细胞,在分选期间使用硅化试管,将细胞沉淀(100×g 3分钟),用500ml PBS洗涤并在旋转轮(12rpm)上在1ml PBS中交联。
    1. (可选)如果需要双交联,首先在室温下用PBS中的2mM Di(N-琥珀酰亚胺基)戊二酸酯交联45分钟,然后轻轻摇动,然后用PBS洗涤两次。
    2. 用1%甲醛在PBS中交联10分钟,同时轻轻摇动
    3. 用125mM甘氨酸淬灭固定,并在摇床上保持5-10分钟
    4. 用冷PBS洗涤两次,在最后一次洗涤用移液管尽可能多的PBS。
    5. 直接在平板上用100μl裂解缓冲液/100,000个细胞裂解细胞(,最少300μl以得到一个输入,一个模拟和一个ChIP样品;400μl,如果2种不同的蛋白质免疫沉淀)。 />
    6. 用细胞刮刀收集细胞,并转移到硅化管中 可选:在液氮中快速冷冻(某些抗体不需要此步骤,如组蛋白标记的抗体)。
    7. 离开在冰上解冻。
    8. 在水浴超声仪中用冰最多超声10分钟(见步骤A3)
    9. 在4℃下以16,000×g离心5分钟,并将上清液转移到新的硅化管中。
    10. 取等分试样(〜10μl),并在1%琼脂糖凝胶上运行以检查剪切(参见图3的最佳结果)。
      注意:确保染色质在500-1,000 bp之间,然后再继续或超声处理。
    11. 在4℃下在旋转轮上用10μl洗涤的Dynabeads /100μl染色质溶液预清洁1小时。
    12. 捕获磁铁上的珠,收集上清液到新的硅化管
    13. 等分100微升在硅化管的输入,模拟ChIP和每个ChIP样品。
    14. 向模拟和ChIP样品中加入400μlChIP IP缓冲液(SDS浓度必须降低至0.1%)。
      注意:请将输入样品保存在-20°C直到第二天。
    15. 向稀释的模拟和ChIP样品中加入正确量的抗体,并在4℃下在轮子上旋转过夜 注意:将剩余的洗过的珠子放在滚轮上过夜,使缓冲液中含有的BSA完全饱和。
    16. 第二天早上,加入25μl饱和Dynabeads ®,并在4°C下放回车轮2小时。
    17. 在磁铁上捕获Dynabeads ,取出上清液并加入1ml冰冷的LiCl洗涤缓冲液。 在室温下放置车轮3-5分钟,重复4次。
    18. 用1ml冰冷的TE缓冲液洗涤1次。 在放置磁铁之前尽快离心,尽可能地除去TE缓冲液。
    19. 通过向洗涤的Dynabeads 直接加入50μl洗脱缓冲液洗脱抗体/染色质复合物。
    20. 短暂涡旋。
    21. 在室温下在热混合器上以1000rpm摇动至少15分钟
    22. 在顶台离心机中以最大速度离心3分钟。
    23. 将管放在磁铁上,收集上清液到新鲜的硅化管中
    24. 重复洗脱步骤,并在同一管中合并洗脱液。
    25. 解冻输入样品,并按照所有样品的后续步骤
    26. 加入10μl5M NaCl(终浓度0.45μM)
    27. 在干热块中煮沸样品15分钟以逆转交联。
    28. 在室温下以最大速度旋转管30秒
    29. 在加入1μl无DNA酶的RNase A之前,让管冷却
    30. 在37℃下孵育15分钟。
    31. 加入0.5μl蛋白酶K.
    32. 在67℃孵育15分钟。
    33. 用低DNA量的试剂盒(例如,DNA MinElute试剂盒)纯化DNA。
    34. 通过与使用实时PCR系统和基于SYBR green的试剂盒进行定量的系列稀释的输入DNA产生的标准曲线进行比较,计算免疫沉淀DNA的量PCR(Frank等人,2001)。作为指导,我们使用每个反应1-2微升纯化的DNA,取决于目标蛋白和要分析的基因数量。沉淀的DNA的最终浓度根据靶蛋白而变化。在NS-5细胞中,我们获得了大约4ng /μl的内源性转录因子和高达30ng /μl的组蛋白标记。

代表数据


图1.小鼠皮质星形胶质细胞中组蛋白标记H3K9乙酰基与抗体量增加的μChIP。在Pax6启动子区(F:cggtgaaagaagccactagg)中进行实时PCR, R:agggaacacaccaactttcg)和在T细胞受体(TCR)的启动子区域(F:cttacaccccaaacctccaa,R:gggaggatgaggagaaaagg)。


图2.在递增量的抗体的NS-5细胞中过表达的HA标记的神经元素2的μChIP。使用识别Neurogenin2(Neurog2)靶基因的启动子区域的引物进行实时PCR (F:gtctcaggaccttcacagtag,R:gagcaaccttctccgtagtag)结合的Fbxw7(F:gcgctttgaagaaagacctg,R:gttttcaaggggcgaatgta)和ORF区域。
图3.在500μl裂解缓冲液中超声处理500,000星形胶质细胞10分钟(30秒开/30秒关闭)之后的最佳染色质溶液,设置H,Diagenode Bioruptor。运行两个样品(各10μl) 在1%琼脂糖凝胶上与DNA梯子一起。

食谱

注意:在开始之前准备储备溶液中所有的新鲜溶液,并保持冰冻。

  1. ChIP IP缓冲区
    20 mM HEPES(pH 7.6)从1 M储备溶液中取出 来自5M储备溶液的0.2M NaCl 2mM EDTA(pH8.0)从0.5M储备液中洗涤 0.1%Na-DOC,来自10%新鲜溶液 1%Triton X-100,来自10%新鲜溶液 5mg/ml BSA,来自250mg/ml储备溶液
    补充蛋白酶抑制剂鸡尾酒
  2. 裂解缓冲液
    0.5%SDS从10%储备溶液中洗涤
    10mM EDTA(pH8.0)从0.5M储备溶液中取出 50 mM Tris-HCl(pH 8.0)从1 M储备溶液中取出 补充1x蛋白酶抑制剂鸡尾酒
  3. 氯化锂(LiCl)洗涤缓冲液
    50mM HEPES(pH 7.6)从1μM储备溶液中取出 1mM EDTA(pH8.0)从0.5M储备溶液中取出 1%NP-40,来自10%新鲜溶液 来自10%新鲜溶液的1%Na-DOC 0.5M LiCl,来自8M储备溶液
  4. Tris-EDTA(TE)缓冲液
    10mM Tris-HCl(pH8.0)从1μM储备溶液中取出1mM EDTA(pH8.0)从0.5M储备溶液中取出
  5. 洗脱缓冲液
    50mM NaHCO 3(pH 5.2)从3M储备溶液中取出 1%SDS从10%储备溶液

致谢

这项工作是由来自Wellcome Trust(WT091800MA)的项目赠款和来自医学研究委员会(U117570528)到FG的授权赠款支持的。该方案使用来自Peggy Farnham的实验室的方法和由FrançoisGuillemot的实验室公布的方法建立 。 我们非常感谢Oishi大夫博士对稿件进行批判性阅读。

参考文献

  1. Frank,SR,Schroeder,M.,Fernandez,P.,Taubert,S.and Amati,B。(2001)。  c-Myc与染色质的结合介导有丝分裂原诱导的组蛋白H4的乙酰化和基因激活。 Genes Dev 15 ):2069-2082。
  2. Masserdotti,G.,Gillotin,S.,Sutor,B.,Drechsel,D.,Irmler,M.,Jorgensen,HF,Sass,S.,Theis,FJ,Beckers,J.,Berninger,B.,Guillemot, F.和Gotz,M。(2015)。 推理因素和REST的调节神经元重编程星形胶质细胞的转录机制。 Cell Stem Cell 17(1):74-88。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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