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ChIP-Seq in Candida albicans
白色念珠菌的染色质免疫共沉淀测序(ChIP-Seq)   

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Abstract

Systems biology approaches can be used to study the regulatory interactions occurring between many components of the biological system at the whole-genome level and decipher the circuitries implicated in the regulation of cellular processes, including those imparting virulence to opportunistic fungi. Candida albicans (C. albicans) is a leading human fungal pathogen. It undergoes morphological switching between a budding yeast form and an elongated multicellular hyphal form. This transition is required for C. albicans’ ability to cause disease and is regulated through highly interconnected regulatory interactions between transcription factors (TFs) and target genes. The chromatin immunoprecipitation (ChIP)-High-throughput sequencing (Seq) technology (ChIP-Seq) is a powerful approach for decoding transcriptional regulatory networks. This protocol was optimized for the preparation of ChIP DNA from filamenting C. albicans cells followed by high-throughput sequencing to identify the targets of TFs that regulate the yeast-to-hyphae transition.

Keywords: Candida albicans(白色念珠菌), Chromatin immunoprecipitation(染色质免疫沉淀), Transcriptional regulatory networks(转录调控网络), Transcription factors(转录因子), Systems biology(系统生物学)

Materials and Reagents

  1. C. albicans strains expressing or not a functional epitope-tagged transcription factor grown under filamentation-inducing conditions in liquid medium [50 ml, e.g. Lee’s medium at 37 °C (Lee et al., 1075)]
  2. 37% formaldehyde (Sigma-Aldrich, catalog number: F8775 )
  3. Liquid nitrogen
  4. Dynabeads® Pan Mouse IgG (5 ml) (Life Technologies, catalog number: 11041 )
  5. Appropriate mouse monoclonal antibody directed against the epitope tag fused to TF [e.g. mouse monoclonal anti-HA antibody, HA-probe Antibody (F-7), Santa Cruz, catalog number: sc-7392 ]
  6. 100 mM Phenylmethylsulfonyl fluoride (PMSF) dissolved in isopropanol (100x stock solution)
  7. DNase-free RNase A solution (10 mg/ml) (Thermo Fisher Scientific, catalog number: EN0531 )
  8. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: B4287 )
  9. Proteinase K solution (20 mg/ml) (Life Technologies, catalog number: AM2546 )
  10. Glycogen (20 mg/ml) (Thermo Fisher Scientific, catalog number: FERR0561 )
  11. Phenol: Chloroform: Isoamyl alcohol (25: 24: 1) (Sigma-Aldrich, catalog number: P2069 )
  12. 5 M NaCl solution
  13. 70%, 100% freezer-cold Ethanol
  14. Quant-iTTM PicoGreen® dsDNA Assay Kit (Life Technologies, catalog number: P11496 )
  15. TruSeqTM DNA Sample Preparation Kit v.2 (Illumina, catalog numbers: FC-121-2001 , FC-121-2002 )
  16. TruSeqTM DNA Sample Preparation Guide v.2 (Illumina, catalog number: FC-930-1021)
  17. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28104 )
  18. MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
  19. E-Gel® iBaseTM and E-Gel® Safe ImagerTM Combo Kit (Life Technologies, catalog number: G6465 ‎)
  20. 2% E-Gel® SizeSelectTM Agarose Gels (Life Technologies, catalog number: G6610-02 )
  21. Glycine (Sigma-Aldrich, catalog number: 50046 )
  22. SDS (Sigma-aldrich, catalog number: L3771 )
  23. 2.5 M glycine (see Recipes)
  24. 10% SDS (see Recipes)
  25. Lee’s medium (see Recipes)
  26. TBS buffer (see Recipes)
  27. Lysis buffer (see Recipes)
  28. PBS/0.5% BSA (see Recipes)
  29. Wash buffer (see Recipes)
  30. Protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11697498001 ) (see Recipes)
  31. TE solution (see Recipes)
  32. TE/SDS solution (see Recipes)

Equipment

  1. 15 ml, 50 ml Falcon tubes
  2. Racks, including a 50-ml Falcon tube rack (Unwire test tube rack, for 30 mm tubes; holds 24) (Thermo Fisher Scientific, Nalgene®, catalog number: 14-809-30 )
  3. VSR-50 laboratory platform rocker (Pro Scientific, catalog number: PSI-512000-00 ) or equivalent
  4. Waste container
  5. 1.5 ml screw-cap tubes
  6. 0.5 ml PCR tube
  7. FastPrep®-24 instrument (MP Biomedicals, catalog number: 116004500 )
  8. Microscope (e.g. Zeiss Axiostar Plus)
  9. 18 G x 1½ inch needles (BD Biosciences, catalog number: 305196 )
  10. 2 ml Eppendorf conical tubes (Eppendorf, catalog number: 022363352 )
  11. Probe sonicator (e.g. MSE Soniprep 150 Plus, exponential microprobe, end diameter 3 mm) [MSE (UK), catalog number: 38121-114A ]
  12. Glass beads (0.5 mm diameter) (Bio Spec Products, catalog number: 11079105 )
  13. Hematology/chemistry mixer 346 (Thermo Fisher Scientific, catalog number: 14-059-346 )
  14. DynaMag Spin magnet system (Life Technologies, model: 123-20D )
  15. Fluorescence reader (e.g. Tecan Trading AG, Infinite®, model: M200 )
  16. BioAnalyzer 2100 (Agilent)
  17. HiSeq 2000 sequencer (Illumina)

Software

  1. Galaxy NGS data analysis software (https://main.g2.bx.psu.edu/)
  2. ChIP-Seq (MACS) peak-finding algorithm software (http://liulab.dfci.harvard.edu/MACS/)

Procedure

  1. Protein-DNA crosslinking
    1. Start a preculture of both tagged and untagged (control) C. albicans strains in 10 ml of rich medium (e.g. Yeast Peptone Dextrose) - allow to grow overnight under vigorous shaking (200 rpm) at 30 °C.
    2. Dilute cells to an optical density at 600 nm (OD600nm) of 0.4 in 50 ml of an appropriate filamentation-inducing liquid medium (e.g. Lee’s medium at 37 °C) and allow to grow under vigorous shaking (200 rpm) until cells reach the equivalent of OD600nm = 1.0 (or typically during 4 h in Lee’s medium at 37 °C).  
    3. Add 1.4 ml of 37% formaldehyde to a 50 ml Falcon tube and transfer the culture (48.6 ml) to the Falcon tube. Screw the caps tightly to prevent leakage of formaldehyde-containing liquid.
    4. Place the Falcon tubes in a suitable rack and tape them. Place the rack on the rocker and agitate during 30 min at room temperature (agitation at 45 rpm using the VSR-50 laboratory platform rocker).
    5. Add 2.5 ml of 2.5 M glycine to stop the cross-linking reaction and incubate 10 min with agitation on the rocker.
    6. Centrifuge the samples for 5 min at 3,500 rpm (2,465 x g). Discard the supernatant (containing formaldehyde) in a suitable waste container. Wash the cells 3 times with 10 ml TBS buffer, by agitating, quickly (1-2 min) centrifuging at 3,500 rpm (2,465 x g) and resuspending in 10 ml TBS buffer.  
    7. Centrifuge the samples for 5 min at 3,500 rpm (2,465 x g) and discard the supernatant. Using the remaining liquid, resuspend the cell pellets and transfer to a 1.5 ml screw-cap tube.
    8. Briefly centrifuge the samples and remove the remaining supernatant. Snap-freeze samples in liquid nitrogen. Store tubes at -80 °C until use.

  2. Preparing Dynabeads for coupling to antibody (work on ice or in a 4 °C room)
    1. Coupling of Dynabeads to antibody for immunoprecipitation (IP) is made the day preceding the scheduled day for preparing extracts. The main stock of beads is vortexed to resuspend the beads and 50 µl of beads per IP are used.
      1.  Transfer the required volume of beads to a 15 ml Flacon tube (e.g. for 6 samples, transfer ~300 µl).
      2. Centrifuge quickly and remove the supernatant.
      3. Wash the beads with 5 ml PBS/0.5% BSA (make a fresh solution, e.g. 0.25 g BSA in 50 ml PBS).
      4. Typically 2 µg of antibody per IP are needed for coupling to the beads at a dilution of 1/10. Resuspend the beads in appropriate volume of PBS/0.5% BSA and add the required volume of antibody at a 1/10 dilution (e.g. for 6 samples and an antibody stock solution at 0.2 µg/µl, transfer 60 µl of antibody to 600 µl of beads resuspended in PBS/0.5% BSA).
      5. Incubate the beads-antibody overnight at 4 °C on a hematology/chemistry mixer.
      6. Quickly centrifuge the 15-ml Falcon tube. Wash the beads-antibody with PBS/BSA 0.5% and resuspend in 30 µl lysis buffer per IP (for 6 samples, resuspend in ~180 µl of lysis buffer).

  3. Preparing total extracts and DNA shearing (work on ice or in a 4 °C room)
    1. Thaw the cell pellets from step 8 on ice and resuspend them in 700 µl of lysis buffer supplemented with protease inhibitor cocktail and 1 mM PMSF (1x final each).
    2. Add the equivalent of 0.5 ml PCR tube of glass beads.
    3. Prepare total cell extracts by bead beating using a FastPrep-24 instrument with 6 runs during 1 min each at 6.0 m/sec and 1 min on ice in between (These settings led to efficient breakage of hyphal cells.).
    4. Make sure of efficient cell breakage by examining 5 µl of the FastPrep processed sample under a light microscope.
    5. Punch a hole at the bottom of the screw cap tube containing the total extracts + beads using an 18 G x 1½ inch needle and quickly place it on a clean 2 ml conical tube. The bottom part of the 1.5 ml screw-cap tube carrying the beads + total extracts has to be introduced into the 2 ml microtube.
    6. Quickly centrifuge (spinning pulse from 0 to ~9,000 rpm) the assembled tubes to collect the total extract from the 1.5 screw cap tube in the 2 ml collection tube. Make sure there are no remaining cells adhered to the glass beads in the 1.5 ml screw cap tube.
    7. Resuspend the collected cell debris + extracts in the 2 ml collection tube and transfer to a clean 1.5 ml microtube.
    8. Sonicate the extracts 4 times during 20 sec at power 8 (knob position) for an output signal amplitude of 21 (Microns, Peak to Peak) using a probe sonicator. Try to get ~200 to 300 bp DNA fragments on average (Figure 1).


      Figure 1. Optimization of DNA shearing by sonication. Total extracts from step 16 (~700 µl) are sonicated 4 times during 20 sec each with 1 min incubation on ice in-between. 4 different sonication power settings [top; 1, knob position 2 output power 6 (Micron, Peak to Peak); 2, knob position 4 output power 10.5; 3, knob position 6 output power 15; 4, knob position 8 output power 21] are tested to optimize DNA fragment size range. Condition 4 is the optimal DNA fragment size range for high-throughput sequencing (~100 bp to maximum 500 bp, maximum average size ~300 bp) DNA ladder sizes are indicated at the left of the panel (bp, base pairs).

    9. Centrifuge the sonicated samples at 14,000 rpm during 10 min at 4 °C and transfer the supernatant (This is the whole cell extract, WCE.) to a clean 1.5 ml microtube.

  4. Chromatin immunoprecipitation (work on ice or in a 4 °C room)
    1. Transfer 500 µl of the WCE to a new 1.5 ml microtube and add 30 µl of Dynabeads coupled to antibody from step B9f.
    2. Incubate the Dynabeads-antibody-WCE mixture overnight at 4 °C under rotation on a hematology/chemistry mixer.

  5. Washing and reversing crosslinks
    1.  In a cold room (4 °C), place the samples on the DynaMag Spin magnet system to allow separation of the Dynabeads from the WCE. Wash twice with 1 ml lysis buffer, twice with 1 ml lysis buffer supplemented with 360 mM NaCl, twice with 1 ml wash buffer and once with 1 ml TE buffer. For each washing step, put the tubes on the magnet, rotate the magnet + tubes 5-6 times to allow efficient attraction of beads, discard the supernatant, add 1 ml of respective buffer, remove the magnet and vigorously agitate the tubes 30-50 times.  
    2. Centrifuge during 2 min at 4,000 rpm at 4 °C and remove the supernatant.
    3. Add 100 µl of TE/SDS solution to the bead pellet. Vortex gently (medium speed) to resuspend and incubate overnight at 65 °C. Vortex for 1 min before leaving at night and vortex again the following morning.  Incubate for another 30 min at 65 °C before proceeding.

  6. DNA purification
    1.  Vortex again at medium speed and incubate during 30 min at 65 °C.
    2. Centrifuge at 14,000 rpm during 2 min and transfer the supernatant to a new 1.5 ml microtube.
    3. Add a mixture of 295 µl TE, 3 µl of RNase A (stock at 10 mg/ml) and 2 µl of glycogen (from stock at 20 mg/ml). Vortex to mix the samples (400 µl final) and incubate at 37 °C during 2 h.
    4. Add 15 µl of 10% SDS and 7.5 µl of proteinase K solution. Vortex gently and incubate 2 h at 37 °C.
    5. Extract twice with 400 µl phenol:chloroform:isoamyl alcohol (25:24:1) by vortexing for 1 min and centrifuging at 14,000 rpm during 10 min at room temperature. Transfer the supernatant from each extraction step to a clean 1.5 ml microtube.
    6. Add 16 µl of 5 M NaCl to the extracted supernatant from step F28 (should be ~350 µl). Vortex then add 2.5 volumes of freezer-cold ethanol (~875 µl). Stored overnight at -20 °C.
    7. Centrifuge at 14,000 rpm during 40 min at 4 °C.
    8. Remove the supernatant and wash with 1 ml of freezer-cold 70% ethanol.
    9. Resuspend the pellet in a convenient volume of H2O (typically 50 µl).
    10. Quantify the IP DNA using the Quant-iTTM PicoGreen® dsDNA Assay Kit following the manufacturer’s instructions.

  7. DNA library generation and high-throughput sequencing
    1. Use 10 ng of IP DNA for library generation. Generate the Illumina library using the TruSeq DNA sample preparation kit v.2 as recommended by the TruSeq DNA sample preparation v.2 guide. Steps include (with some modifications):
      1. End repair as recommended in TruSeq DNA sample preparation v.2 guide (page 43).
      2. Purification of end-repaired DNA with QIAquick PCR Purification kit as recommended by the manufacturer, instead of using Agencourt AMPure XP beads.
      3. Adenylation of 3’ DNA ends as recommended in TruSeq DNA sample preparation v.2 guide (page 47).
      4. Ligation of indexed adapters as recommended in TruSeq DNA sample preparation v.2 guide (page 49).
      5. Clean up with Qiagen MinElute PCR purification kit as recommended by the manufacturer, instead of using Agencourt AMPure XP beads.
      6. Use E-Gel® iBaseTM and E-Gel® Safe ImagerTM Combo Kit and E-Gel® SizeSelectTM Agarose Gels, 2% following the instructions provided by the manufacturer to purify the desired library fragments, instead of using 2% agarose with SyBr Gold gel. Migrate samples on E-gel during 20 min as recommended by Invitrogen. Select fragments with an average size of ~300 bp.    
      7. Enrich DNA fragments by amplification with PCR for 10 cycles as recommended in TruSeq DNA sample preparation v.2 guide (page 58).
      8. Clean up PCR with QIAquick PCR Purification kit as recommended by the manufacturer, instead of using Agencourt AMPure XP beads.
      9. Validate library and perform quality control by loading and migrating DNA on a BioAnalyzer 2100 device with a High Sensitivity DNA chip (Figure 2) following the manufacturer’s instructions and as indicated on page 62 of the TruSeq DNA sample preparation v.2 guide. 


        Figure 2. Quality control of purified Illumina adapter-ligated DNA library using the BioAnalyzer 2100. Representative BioAnalyzer 2100 migration profile of Illumina high-throughput sequencing DNA library from ChIP assay (Library). Illumina adapter dimers are indicated (Adapter dimer). DNA fragment sizes are indicated on the x-axis [(bp), base pairs] and DNA abundance on the y-axis [(FU), fluorescence units].

    2. Dilute ChIP library samples to 2 nM, denature DNA samples and load onto an Illumina HiSeq 2000 sequencer flow cell lane for single-read (51 base pairs per read) high-throughput sequencing following the manufacturer’s instructions.
    3. The resulting read files (FASTQ) are pre-processed with the online Galaxy NGS data analysis software (Blankenberg et al., 2010; Giardine et al., 2005). Steps include:
      1. Quality control analyses of the FASTQ files using FastQC version 0.52 on the Galaxy web interface - proceed to clipping of adapter-contaminated sequences using Clip version 1.0.1.
      2. Mapping of the resulting files to the C. albicans assembly 21 genome (http://www.candidagenome.org/download/chromosomal_feature_files/C_albicans_SC5314/) using the Bowtie algorithm (Galaxy: Click on NGS mapping>Map with Bowtie for Illumina version 1.1.2) - the Assembly 21 of the C. albicans genome can be imported into Galaxy by clicking on User>Custom builds (top window of the Galaxy web interface). Mapping will generate BAM files for processing with peak finding algorithms.
    4. Proceed to peak finding with the generated BAM files using the Model-Based Analysis for ChIP-Seq (MACS) peak-finding algorithm software and following the instructions found on one of the two corresponding detailed protocols (Feng et al., 2012; Feng et al., 2011).

Recipes

  1. 2.5 M glycine
    Dissolve 93.8 g of glycine in 500 ml H2O
  2. 10% SDS
    Dissolve 10 g of SDS in 100 ml H2O
  3. Lee’s medium (for 1 L)
    5.0 g of (NH4)2SO4
    0.2 g of MgSO4.7H2O
    2.5 g of K2HPO4
    5.0 g of NaCl
    12.5 g of D-Glucose
    0.5 g of L-Alanine
    1.3 g of L-Leucine
    1.0 g of L-Lysine
    0.1 g of L-Methionine
    0.0714 g of L-Ornithine
    0.5 g of L-Phenylalanine
    0.5 g of L-Proline
    0.5 g of L-Threonine
    0.001 g of Biotin
    Distilled water to 1 L
    Combine ingredients (except biotin) and autoclave at 110 °C for 20 min
    Add biotin
    pH 6.8 ± 0.05
  4. 5x TBS buffer (for 1 L)
    100 ml of 1M Tris-HCl (pH 7.5) (100 mM final at 5x)
    150 ml of 5M NaCl (750 mM NaCl final at 5x)
    H2O to 1 L (750 ml)
    Filter sterilize
    Stored at 4 °C
  5. 1x lysis buffer (for 500 ml)
    25 ml of 1M HEPES-KOH (pH 7.5) (50 mM final at 1x)
    14 ml of 5M NaCl (140 mM final at 1x)
    1 ml of 500 mM EDTA (1 mM final at 1x)
    50 ml of 10% Triton X100 (1% final at 1x)
    10 ml of 5% Na-deoxycholate (0.1% final at 1x)
    H2O to 500 ml (400 ml)
    Filter sterilize
    Stored at 4 °C
  6. 1x PBS/0.5% BSA (for 50 ml)
    Preparation of 1x PBS solution
    1 L at 1x: To ~800 ml of H2O add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4 and stirr with a magnetic stirrer. Adjust the pH to 7.4 with HCl and complete with H2O to 1 L. Stored at room temperature.
    Preparation of PBS/0.5% BSA
    Add 0.25 g of Bovine Serum Albumin to 50 ml 1x PBS in a 50-ml Falcon tube. Do not mix, simply stored at 4 °C for ~ 30 min to 1 h.
  7. 1x wash buffer (for 500 ml)
    5 ml of 1 M Tris-HCl (pH 8.0) (10 mM final at 1x)
    25 ml of 5 M LiCl (250 mM final at 1x)
    2.5 ml of 100% NP40 (0.5% final at 1x)
    25 ml of 10% Na-deoxycholate (0.5% final at 1x)
    1 ml of 500 mM EDTA (1mM final at 1x)
    H2O to 500 ml (441.5 ml)
  8. Protease inhibitor cocktail tablets
    25x stock solution made by dissolving one tablet in 2 ml H2O
    Aliquot 100 µl in 1.5 ml tubes
    Stored at -20 °C
  9. TE solution
    10 mM Tris (pH 8.0)
    1 mM EDTA
  10. TE/SDS solution
    10 mM Tris (pH 8.0)
    1 mM EDTA
    1% sodium dodecyl sulfate

Acknowledgments

This protocol was adapted from Drouin & Robert « Genome-wide Location Analysis of Chromatin-associated Proteins by ChIP on CHIP: Controls Matter » available at : http://www.ircm.qc.ca/LARECHERCHE/axes/Biologie/Chromatine/Documents/ProtocoleIRCM_LevureYeast1.pdf and Znaidi et al. (2013). This work was supported by grants from the European commission (FinSysB PITN-GA-2008-214004), the Agence Nationale de la Recherche (KANJI, ANR-08-MIE- 033-01) and a BIOASTER-Sanofi-Alliance pour les Sciences de la Vie et de la Santé (AVIESAN) joint program to Dr. Christophe d’Enfert.

References

  1. Blankenberg, D., Von Kuster, G., Coraor, N., Ananda, G., Lazarus, R., Mangan, M., Nekrutenko, A. and Taylor, J. (2010). Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol Chapter 19: Unit 19 10 11-21.
  2. Feng, J., Liu, T. and Zhang, Y. (2011). Using MACS to identify peaks from ChIP-Seq data. Curr Protoc Bioinformatics Chapter 2: Unit 2 14.
  3. Feng, J., Liu, T., Qin, B., Zhang, Y. and Liu, X. S. (2012). Identifying ChIP-seq enrichment using MACS. Nat Protoc 7(9): 1728-1740.
  4. Giardine, B., Riemer, C., Hardison, R. C., Burhans, R., Elnitski, L., Shah, P., Zhang, Y., Blankenberg, D., Albert, I., Taylor, J., Miller, W., Kent, W. J. and Nekrutenko, A. (2005). Galaxy: a platform for interactive large-scale genome analysis. Genome Res 15(10): 1451-1455.
  5. Lee, K. L., Buckley, H. R. and Campbell, C. C. (1975). An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida Albicans. Sabouraudia 13(2): 148-153.
  6. Znaidi, S., Nesseir, A., Chauvel, M., Rossignol, T. and d'Enfert, C. (2013). A comprehensive functional portrait of two heat shock factor-type transcriptional regulators involved in Candida albicans morphogenesis and virulence. PLoS Pathog 9(8): e1003519.

简介

系统生物学方法可以用于研究在全基因组水平上发生在生物系统的许多组分之间的调节相互作用,并且解释涉及细胞过程调节的电路,包括赋予机会性真菌的毒力的电路。 白色念珠菌( C。albicans )是一种主要的人类真菌病原体。它经历萌芽酵母形式和细长多细胞菌丝形式之间的形态学转换。此转换是 C所必需的。白色念珠菌引起疾病的能力,并且通过转录因子(TF)和靶基因之间高度相互关联的调节相互作用来调节。染色质免疫沉淀(ChIP) - 高通量测序(Seq)技术(ChIP-Seq)是解码转录调节网络的强有力的方法。该方案针对从丝状结构C制备ChIP DNA进行了优化。白色念珠菌细胞,然后进行高通量测序以鉴定调节酵母对菌丝转变的TF的靶标。

关键字:白色念珠菌, 染色质免疫沉淀, 转录调控网络, 转录因子, 系统生物学

材料和试剂

  1. C。 在液体培养基(50ml,例如Lee's培养基,37℃)中在长丝诱导条件下生长的表达或不表达功能性表位标记的转录因子的白色念珠菌菌株(Lee等人 。,1075)]
  2. 37%甲醛(Sigma-Aldrich,目录号:F8775)
  3. 液氮
  4. Dynabeads Pan Mouse IgG(5ml)(Life Technologies,目录号:11041)
  5. 适合的针对与TF [例如小鼠单克隆抗HA抗体,HA探针抗体(F-7),Santa Cruz,目录号:sc-7392]融合的表位标签的小鼠单克隆抗体
  6. 100mM苯甲基磺酰氟(PMSF)溶于异丙醇(100×储备液)中
  7. 无DNA酶的RNase A溶液(10mg/ml)(Thermo Fisher Scientific,目录号:EN0531)
  8. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:B4287)
  9. 蛋白酶K溶液(20mg/ml)(Life Technologies,目录号:AM2546)
  10. 糖原(20mg/ml)(Thermo Fisher Scientific,目录号:FERR0561)
  11. 苯酚:氯仿:异戊醇(25:24:1)(Sigma-Aldrich,目录号:P2069)
  12. 5 M NaCl溶液
  13. 70%,100%冷冻乙醇
  14. ds-DNA测定试剂盒(Life Technologies,目录号:P11496)。
  15. TruSeq DNA样品制备试剂盒v.2(Illumina,目录号:FC-121-2001,FC-121-2002)
  16. TruSeq TM DNA样品制备指南v.2(Illumina,目录号:FC-930-1021)
  17. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  18. MinElute PCR纯化试剂盒(QIAGEN,目录号:28004)
  19. E-Gel iBase TM 和E-Gel Safe Imager TM Combo Kit :G6465)
  20. 2%E-Gel SizeSelect TM 琼脂糖凝胶(Life Technologies,目录号:G6610-02)
  21. 甘氨酸(Sigma-Aldrich,目录号:50046)
  22. SDS(Sigma-aldrich,目录号:L3771)
  23. 2.5 M甘氨酸(参见配方)
  24. 10%SDS(见配方)
  25. 李的媒介(见配方)
  26. TBS缓冲区(请参阅配方)
  27. 裂解缓冲液(见配方)
  28. PBS/0.5%BSA(参见配方)
  29. 洗涤缓冲液(见配方)
  30. 蛋白酶抑制剂混合物片剂(Roche Diagnostics,目录号:11697498001)(参见Recipes)
  31. TE解决方案(参见配方)
  32. TE/SDS溶液(参见配方)

设备

  1. 15 ml,50ml Falcon管
  2. 机架,包括50-ml Falcon管架(Unwire试管架,用于30mm管;保持24)(Thermo Fisher Scientific,Nalgene ,目录号:14-809-30)
  3. VSR-50实验室平台摇杆(Pro Scientific,目录号:PSI-512000-00)或等同物
  4. 垃圾箱
  5. 1.5 ml螺旋盖管
  6. 0.5 ml PCR管
  7. FastPrep -24仪器(MP Biomedicals,目录号:116004500)
  8. 显微镜(例如 Zeiss Axiostar Plus)
  9. 18 G×1/2英寸针(BD Biosciences,目录号:305196)
  10. 2ml Eppendorf锥形管(Eppendorf,目录号:022363352)
  11. 探针超声波仪(例如MSE Soniprep 150 Plus,指数微探针,端径3mm)[MSE(UK),目录号:38121-114A]
  12. 玻璃珠(直径0.5mm)(Bio Spec Products,目录号:11079105)
  13. 血液/化学混合器346(Thermo Fisher Scientific,目录号:14-059-346)
  14. DynaMag自旋磁体系统(Life Technologies,型号:123-20D)
  15. 荧光阅读器(例如Tecan Trading AG,Infinite ,型号:M200)
  16. BioAnalyzer 2100(Agilent)
  17. HiSeq 2000 sequencer(Illumina)

软件

  1. Galaxy NGS数据分析软件( https://main.g2.bx.psu.edu/
  2. ChIP-Seq(MACS)峰值寻找算法软件( http://liulab.dfci.harvard.edu/MACS/

程序

  1. 蛋白质-DNA交联
    1. 开始标记和未标记(对照)的预培养。 在10ml富含培养基(例如酵母蛋白胨右旋糖)中的白色念珠菌菌株 - 允许   在30℃下在剧烈摇动(200rpm)下生长过夜
    2. 将细胞稀释至在50ml中的600nm(OD 600nm)的光密度为0.4 的适当的细胞诱导液体培养基(例如Lee的 培养基在37℃),并允许在剧烈振荡(200rpm)下生长, 直到细胞达到OD 600nm = 1.0的等效值(或通常在4天内)   h在Lee's培养基中37℃)。  
    3. 加入1.4ml 37%甲醛 加入到50ml Falcon管中,并将培养物(48.6ml)转移到Falcon 管。 拧紧盖子以防止泄漏 含甲醛液体
    4. 将猎鹰管放在 合适的机架和胶带。 将机架放在摇杆上并搅拌 在室温下30分钟(使用VSR-50以45rpm搅拌) 实验室平台摇杆)。
    5. 加入2.5ml 2.5M甘氨酸以停止交联反应,并在摇动器上摇动孵育10分钟。
    6. 将样品以3500rpm(2,465×g)离心5分钟。 丢弃   上清液(含有甲醛)在合适的废物容器中。 用10ml TBS缓冲液洗涤细胞3次,通过搅拌,快速(1-2   min)以3500rpm(2,465×g)离心并重悬于10ml中 TBS缓冲液。  
    7. 将样品以3500rpm离心5分钟 (2,465×g)并弃去上清液。 使用剩余的液体, 重悬细胞沉淀并转移到1.5ml螺旋盖管
    8. 短暂离心样品并除去剩余的上清液。 在液氮中快速冷冻样品。 将管在-80°C储存,直到使用。

  2. 准备Dynabeads用于偶联到抗体(在冰上或在4℃室中工作)
    1. 进行Dynabeads与用于免疫沉淀(IP)的抗体的偶联 在用于制备提取物的预定日之前的一天。 主要的 涡旋珠子的储液以重悬珠子和每50μl珠子 IP。
      1.  将所需体积的珠子转移到15 ml Flacon管中(例如 6个样品,转移〜300μl)。
      2. 快速离心并除去上清液。
      3. 用5ml PBS/0.5%BSA洗涤珠子(制备新鲜溶液,例如在50ml PBS中的0.25g BSA)。
      4. 通常需要每个IP2μg抗体以偶联至珠粒   稀释1/10。 将珠子以适当体积重悬 PBS/0.5%BSA,并以1/10稀释度加入所需体积的抗体 (例如,对于6个样品和0.2μg/μl的抗体储备溶液) 将60μl抗体转移到600μl重悬于PBS/0.5% BSA)。
      5. 在4℃下,在血液/化学混合器上孵育珠子抗体过夜
      6. 快速离心15毫升Falcon管。 用珠子抗体洗涤   PBS/BSA 0.5%,并重悬于30μl裂解缓冲液/IP(对于6个样品,   重悬于〜180μl裂解缓冲液中)。

  3. 制备总提取物和DNA剪切(在冰上或在4℃室中进行)
    1. 解冻来自步骤8的细胞沉淀在冰上,并将其重悬在700微升 的补充有蛋白酶抑制剂混合物和1mM的裂解缓冲液 PMSF(每次1x决赛)。
    2. 加入相当于0.5ml PCR管的玻璃珠。
    3. 使用FastPrep-24通过珠磨制备总细胞提取物 仪器,6次运行1分钟,每次6.0m /秒,在冰上1分钟 (这些设置导致菌丝细胞的有效破裂。)。
    4. 通过在光学显微镜下检查5μlFastPrep处理的样品,确保有效的细胞破碎
    5. 在包含总量的螺旋盖管的底部打孔 提取+珠使用18 G x1½英寸针,并迅速将其放在   清洁2ml锥形管。 1.5毫升螺旋盖管的底部 携带珠+总提取物必须引入2毫升 微管
    6. 快速离心(纺丝脉冲从0到〜9,000  rpm)组装的管以收集来自1.5的总提取物 螺旋盖管在2ml收集管中。确保没有 剩余的细胞粘附在1.5ml螺旋盖管中的玻璃珠上
    7. 重新悬浮收集细胞碎片+提取物在2毫升收集管,并转移到干净的1.5毫升微管。
    8. 超声提取4次,在20秒的电源8(旋钮位置)  对于21的输出信号幅度(微米,峰值到峰值)使用a 探头超声波仪。尝试平均得到约200到300 bp的DNA片段 (图1)。


      图1.通过超声处理优化DNA剪切。在20天期间将来自步骤16的总提取物(约700μl)超声处理4次 秒,在其间的冰上孵育1分钟。 4种不同的超声波处理 电源设置[顶部; 1,旋钮位置2输出功率6(Micron,Peak to   峰); 2,旋钮位置4输出功率10.5; 3,旋钮位置6输出 功率15; 4,旋钮位置8输出功率21]进行测试以优化DNA   片段大小范围。 条件4是最佳DNA片段大小范围   用于高通量测序(〜100bp至最大500bp,最大 平均大小约300 bp)DNA梯级大小显示在左侧   (bp,碱基对)。

    9. 离心经超声处理的样品   在14,000rpm,在4℃下10分钟,并转移上清液(这 是全细胞提取物,WCE)到干净的1.5ml微量管中。

  4. 染色质免疫沉淀(在冰上或在4℃室中进行)
    1. 转移500微升的WCE到一个新的1.5毫升微管,并加入30微升与步骤B9f抗体耦合的Dynabeads。
    2. 在4℃下,在血液学/化学混合器上旋转,将Dynabeads-抗体-WCE混合物孵育过夜。

  5. 洗涤和逆转交联
    1.  在寒冷的房间(4°C),将样品放在DynaMag Spin磁铁上 系统以允许Dynabeads与WCE分离。 洗两次 用1ml裂解缓冲液,用1ml裂解缓冲液补充两次 360mM NaCl,用1ml洗涤缓冲液洗涤两次,用1ml TE缓冲液洗涤一次。 对于每个洗涤步骤,将管放在磁铁上,旋转磁铁+ 管5-6次以允许有效吸引珠,丢弃 上清液,加入1ml各自的缓冲液,去除磁铁 大力搅拌试管30-50次。  
    2. 在4℃下以4,000rpm离心2分钟,除去上清液
    3. 加入100微升TE/SDS溶液到珠粒。 轻轻涡旋 (中速)重悬并在65℃温育过夜。 漩涡1   分钟,然后在第二天早上再次涡旋。 在65℃下再孵育30分钟,然后进行。

  6. DNA纯化
    1.  以中等速度再次涡旋,并在65℃孵育30分钟。
    2. 在14,000rpm离心2分钟,并将上清液转移到新的1.5ml微量管
    3. 加入295微升TE,3微升RNA酶A(10毫克/毫升的股票)和2 μl的糖原(来自20mg/ml的原液)。 涡旋混合样品(400 μl终浓度)并在37℃孵育2小时。
    4. 加入15μl的10%SDS和7.5μl的蛋白酶K溶液。 轻轻涡旋并在37℃孵育2小时
    5. 用400μl苯酚:氯仿:异戊醇(25:24:1)提取两次, 通过涡旋1分钟并在14,000rpm下离心10分钟 室内温度。 将每个提取步骤的上清液转移到a   清洁1.5ml微管。
    6. 加入16μl的5 M NaCl 从步骤F28提取的上清液(应该〜350μl)。 然后加入Vortex   2.5体积的冷冻的乙醇(〜875μl)。 在-20℃过夜贮存 ℃。
    7. 在4℃下以14,000rpm离心40分钟。
    8. 除去上清液,用1ml冷冻冷却的70%乙醇洗涤
    9. 在合适体积的H 2 O(通常为50μl)中重悬沉淀物
    10. 根据制造商的说明书,使用Quant-iT PicoGreen dsDNA测定试剂盒定量IP DNA。

  7. DNA文库生成和高通量测序
    1. 使用10 ng的IP DNA进行文库生成。 生成Illumina 库,使用TruSeq DNA样品制备试剂盒v.2 通过TruSeq DNA样品制备v.2指南。 步骤包括(有一些   修改):
      1. 按照TruSeq DNA样品制备v.2指南(第43页)中的建议进行末端修复。
      2. 用QIAquick PCR纯化末端修复的DNA纯化试剂盒as   推荐由制造商,而不是使用Agencourt AMPure XP 珠子
      3. 3'DNA末端的腺苷酸化,如TruSeq DNA样品制备v.2指南(第47页)中推荐的。
      4. 按照TruSeq DNA样品制备v.2指南(第49页)中的建议连接索引适配器。
      5. 用Qiagen MinElute PCR纯化试剂盒按照推荐方法清洗 制造商,而不是使用Agencourt AMPure XP珠
      6. 使用E-Gel ® iBase TM 和E-Gel ® Safe Imager TM Combo Kit and E-Gel < sup>® SizeSelect TM 琼脂糖凝胶,2%,按照 制造商来纯化所需的文库片段,而不是使用 2%琼脂糖与SyBr Gold凝胶。 在20分钟期间将样品在E凝胶上迁移   推荐由Invitrogen。 选择平均大小为〜300 bp的片段。    
      7. 通过PCR扩增来富集DNA片段 10个循环,如TruSeq DNA样品制备v.2指南中推荐的 (第58页)。
      8. 用QIAquick PCR纯化试剂盒纯化PCR 推荐由制造商,而不是使用Agencourt AMPure XP 珠子
      9. 验证库并通过加载执行质量控制 并在具有高灵敏度的BioAnalyzer 2100装置上迁移DNA DNA芯片(图2)按照制造商的说明书和as 如TruSeq DNA样品制备v.2指南第62页所示。


        图   纯化的Illumina适配器连接的DNA文库的质量控制 使用BioAnalyzer 2100。代表性BioAnalyzer 2100迁移 简介来自ChIP的Illumina高通量测序DNA文库 测定(Library)。 指示了Illumina适配器二聚体(衔接子二聚体)。 DNA片段大小显示在x轴[(bp),碱基对]和 DNA丰度在y轴[(FU),荧光单位]。

    2. 将ChIP文库样品稀释至2 nM,变性DNA样品并上样   用于单读的Illumina HiSeq 2000测序流通池通道(51 碱基对每读)高通量测序 制造商的说明
    3. 生成的读取文件(FASTQ) 使用在线Galaxy NGS数据分析软件进行预处理 (Blankenberg等人,2010; Giardine等人,2005)。 步骤包括:
      1. 使用FastQC版本0.52对FASTQ文件进行质量控制分析 在Galaxy的网络界面 - 继续剪辑 适配器污染的序列,使用Clip版本1.0.1。
      2. 将生成的文件映射到 C。白色念珠菌组装21基因组 ( http://www.candidagenome.org/download/chromosomal_feature_files/C_albicans_SC5314/)  使用Bowtie算法(Galaxy:Click on NGS mapping> Map with Bowtie for Illumina 1.1.2版) - C的装配21。 albicans 基因组可以通过点击用户>自定义构建导入到Galaxy (Galaxy Web界面的顶部窗口)。映射将生成BAM 用于使用峰值发现算法处理的文件。
    4. 继续 使用基于模型的分析与生成的BAM文件进行峰值查找  用于ChIP-Seq(MACS)峰值寻找算法软件和以下 在两个相应的详细协议之一上找到的指令 (Feng等人,2012; Feng等人,2011)。

食谱

  1. 2.5M甘氨酸 将93.8g甘氨酸溶解在500ml H 2 O中
  2. 10%SDS
    将10克SDS溶于100毫升H 2 O中
  3. 李氏中等(1升)
    将5.0g的(NH 4)2 SO 4 SO 4/
    0.2g MgSO 4 .7H 2 O 2 2.5克K 2 HPO 4
    5.0g NaCl
    12.5克D-葡萄糖 0.5克L-丙氨酸 1.3克L-亮氨酸 1.0g L-赖氨酸 0.1克L-甲硫氨酸 0.0714g L-鸟氨酸 0.5克L-苯丙氨酸 0.5克L-脯氨酸 0.5克L-苏氨酸 0.001克生物素
    蒸馏水至1 L
    将成分(除生物素之外)在110℃下高压灭菌20分钟
    添加生物素
    pH 6.8±0.05
  4. 5x TBS缓冲液(1 L)
    100ml 1M Tris-HCl(pH7.5)(100mM终于5×) 150ml 5M NaCl(750mM NaCl,最终浓度为5×) H 2 O至1L(750ml) 过滤灭菌
    储存在4°C
  5. 1x裂解缓冲液(500 ml)
    25ml 1M HEPES-KOH(pH7.5)(50mM终浓度,1x)
    14ml 5M NaCl(140mM终浓度,1x)
    1ml 500mM EDTA(1mM终浓度为1mM)
    50ml 10%Triton X100(1%终浓度1%) 10ml 5%脱氧胆酸钠(0.1%终浓度,1x)
    H 2 O至500ml(400ml) 过滤灭菌
    储存在4°C
  6. 1×PBS/0.5%BSA(对于50ml) 制备1x PBS溶液
    1L在1×:向〜800ml的H 2 O添加8g的NaCl,0.2g的KCl,1.44g的Na 2 HPO 4水溶液, 0.24g KH 2 PO 4和用磁力搅拌器搅拌。 用HCl调节pH至7.4,用H 2 O至1L完全。在室温下贮存。
    PBS/0.5%BSA的制备 在50ml Falcon管中,将0.25g牛血清白蛋白加入50ml 1x PBS中。 不要混合,简单地储存在4°C〜30分钟到1小时。
  7. 1x洗涤缓冲液(500 ml)
    5ml的1M Tris-HCl(pH8.0)(最终浓度为10mM,1x)
    25ml 5M LiCl(250mM终浓度为1x)
    2.5ml 100%NP40(0.5%最终在1x)
    25ml 10%脱氧胆酸钠(0.5%最终在1x) 1ml 500mM EDTA(1mM终浓度为1x)
    H 2 O至500ml(441.5ml)
  8. 蛋白酶抑制剂鸡尾酒片剂
    通过将一片溶解在2ml H 2 O中制备的25x储备溶液 等分100μl在1.5 ml管
    储存于-20°C
  9. TE解决方案
    10mM Tris(pH8.0) 1mM EDTA
  10. TE/SDS溶液
    10mM Tris(pH8.0) 1mM EDTA
    1%十二烷基硫酸钠

致谢

该方案改编自Drouin& Robert«Genomic-wide Location analysis of Chromatin-associated Proteins by ChIP on CHIP:Controls Matter»available at:http://www.ircm.qc.ca/LARECHERCHE/axes/Biologie/Chromatine/Documents/ProtocoleIRCM_LevureYeast1.pdf和Znaidi 等(2013)。这项工作得到了来自欧洲委员会(FinSysB PITN-GA-2008-214004),Agence Nationale de la Recherche(KANJI,ANR-08-MIE-033-01)和BIOASTER-Sanofi-Alliance pour Sciences de la Vie et de laSanté(AVIESAN)联合项目Christophe d'Enfert博士。

参考文献

  1. Blankenberg,D.,Von Kuster,G.,Coraor,N.,Ananda,G.,Lazarus,R.,Mangan,M.,Nekrutenko,A.and Taylor,J.(2010)。 Galaxy:基于网络的实验员基因组分析工具 Curr Protoc Mol Biol Chapter 19:Unit 19 10 11-21。
  2. Feng,J.,Liu,T.and Zhang,Y.(2011)。 使用MACS从ChIP-Seq数据中识别峰。 Curr Protoc Bioinformatics第 2章:第2章14.
  3. Feng,J.,Liu,T.,Qin,B.,Zhang,Y.and Liu,X.S。(2012)。 使用MACS识别ChIP-seq富集。 Nat Protoc 7(9):1728-1740
  4. Giardine,B.,Riemer,C.,Hardison,RC,Burhans,R.,Elnitski,L.,Shah,P.,Zhang,Y.,Blankenberg,D.,Albert,I.,Taylor, ,W.,Kent,WJ和Nekrutenko,A。(2005)。 Galaxy:用于交互式大规模基因组分析的平台 Genome Res 15(10):1451-1455
  5. Lee,K.L.,Buckley,H.R。和Campbell,C.C。(1975)。 一种氨基酸液体合成培养基,用于发展白色念珠菌的菌丝体和酵母形式/em>。 Sabouraudia 13(2):148-153。
  6. Znaidi,S.,Nesseir,A.,Chauvel,M.,Rossignol,T.and d'Enfert,C.(2013)。 参与白色念珠菌的两种热休克因子型转录调节因子的综合功能描述形态发生和毒力。 PLoS Pathog 9(8):e1003519。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Znaidi, S., Proux, C., Weber, S., Drouin, S., Robert, F., Raymond, M., Coppée, J. and d’Enfert, C. (2014). ChIP-Seq in Candida albicans. Bio-protocol 4(12): e1158. DOI: 10.21769/BioProtoc.1158.
  2. Znaidi, S., Nesseir, A., Chauvel, M., Rossignol, T. and d'Enfert, C. (2013). A comprehensive functional portrait of two heat shock factor-type transcriptional regulators involved in Candida albicans morphogenesis and virulence. PLoS Pathog 9(8): e1003519.
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