Quantitative ChIP-seq by Adding Spike-in from Another Species
通过引入另一物种的标准参照以定量分析ChIP-seq   

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Abstract

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a routine procedure in the lab; however, epigenome-wide quantitative comparison among independent ChIP-seq experiments remains a challenge. Here, we contribute an experimental protocol combined with a computational workflow allowing quantitative and comparative assessment of epigenome using animal tissues.

Keywords: Epigenome (表观基因组), H3K27me3 (H3K27me3), Quantitative ChIP-seq (定量 ChIP-seq), Spike-in (外源参照), Drosophila (果蝇)

Background

Chromatin and epigenetic complexes that modify histones regulate the accessibility of DNA to transcriptional machinery, thereby permitting direct control of gene expression. To characterize the epigenomic feature of histone modification, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become a widely used method. However, traditional ChIP-seq protocols are not inherently quantitative and therefore prohibit direct comparison between samples derived from distinct cell types or cells that have been through different genetic or chemical perturbation. Despite the fact that several in silico normalization methods have been proposed to overcome this disadvantage, an experiment-based strategy is still lacking. In 2014, Orlando et al. (2014) developed a method, called ChIP with reference exogenous genome (ChIP-Rx), which utilizes a constant amount of reference or ‘‘spike-in’’ epigenome for cell-based comparison among epigenomes. In current protocol, we have refined this method by using the percentage of mapped spike-in reference epigenome. And we have successfully applied this protocol in direct comparison between two or more ChIP-seq datasets from animal tissues.

Materials and Reagents

  1. Consumables
    1. Pipette tips
    2. 1.5 ml microcentrifuge tube
    3. 15 ml microcentrifuge tube
    4. 1.5 ml Bioruptor Microtubes (Diagenode, catalog number: C30010016 )
    5. 10 cm Petri-dish
    6. Glass Dounce tube

  2. Biological materials
    1. Mouse Neuro-2a cells
    2. Drosophila (1,000 embryos [30 min-1 h after egg-laying], 30 larvae [96 h after egg-laying], and 30 pupae [7 d after egg-laying])
      Starting material: Cells were cultured at 37 °C, 5% CO2, and saturated humidity in complete LG-DMEM (Thermo Fisher Scientific, Life Technologies, catalog number: 11995-065 ) containing 10% FBS (Sigma-Aldrich, catalog number: 12003C ), seeded at 1.0 x 106 cells/cm2 in a 10 cm Petri-dish. Flies were cultured in standard Drosophila media (Recipe 1) at 25 °C with 60% humidity in a 12 h light and 12 h dark cycle unless otherwise specified.

  3. Reagents
    ChIP reagents
    1. Formaldehyde, 37% (weight/volume) (Sigma-Aldrich, catalog number: 252549 ), stored at room temperature (20-25 °C)
    2. Glycine (Sigma-Aldrich, catalog number: G7403 ), stored at room temperature (20-25 °C)
    3. PBS buffer, 20x (Sangon Biotech, catalog number: B548117 ), stored at room temperature (20-25 °C)
    4. RIPA buffer (Sigma-Aldrich, catalog number: R0278 ), stored at 4 °C
    5. Tris buffer, 1 M, PH 8.0 (Sangon Biotech, catalog number: B548127 ), stored at room temperature (20-25 °C)
    6. Sodium chloride, 5 M (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9759 ), stored at room temperature (20-25 °C)
    7. Triton X-100 (Sigma-Aldrich, catalog number: T8787 ), stored at 4 °C
    8. EDTA, 0.5 M, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9260G ), stored at room temperature (20-25 °C)
    9. SDS, 10% (weight/volume) (Sangon Biotech, catalog number: B548118 ), stored at room temperature (20-25 °C)
    10. cOmplete proteinase inhibitors cocktail tablets (Roche Diagnostics, catalog number: 11697498001 ), stored at 4 °C
    11. Dynabeads Protein G (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10004D ), stored at 4 °C
    12. Anti-trimethyl Histone H3 (Lys27) antibody (Merck, catalog number: 07-449 ), stored at -20 °C
    13. Sodium bicarbonate (Sangon Biotech, catalog number: A100865 ), stored at room temperature (20-25 °C)
    14. Proteinase K (Sangon Biotech, catalog number: A600451 ), stored at -20 °C
    15. RNase A, 10 mg/ml (Thermo Fisher Scientific, catalog number: EN0531 ), Stored at -20 °C
    16. PCR purification kit (QIAGEN, catalog number: 28106 ), stored at room temperature (20-25 °C)
    17. Qubit dsDNA HS assay kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 ), stored at 4 °C (Warning: protect from light)
    18. Trypsin (Thermo Fisher Scientific, catalog number: 25300054 )
    19. Liquid nitrogen
    20. Glycine, 2.5 M (see Recipes)
    21. ChIP Wash Buffer (see Recipes)
    22. ChIP Final Wash Buffer (see Recipes)
    23. ChIP Elution Buffer (see Recipes)

    Library reagents
    1. Agencourt AMPure XP (SPRI beads; Beckman Coulter, catalog number: A63881 ), stored at 4 °C
    2. NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, catalog number: E7370L ), stored at -20 °C
    3. NEBNext Multiplex Oligos for Illumina (New England Biolabs, catalog number: E7335L ), stored at -20 °C
    4. Gel extraction kit (QIAGEN, catalog number: 20021 ), stored at room temperature (20-25 °C)
    5. Sodium hydroxide solution, 10 M (Sigma-Aldrich, catalog number: 72068 )

Equipment

  1. Bioruptor Pico (Diagenode)
  2. DynaMag-2 magnet (Thermo Fisher Scientific, catalog number: 12321D )
  3. Mortar and pestle
  4. 1 ml glass homogenizer
  5. Water bath
  6. Rotate
  7. Centrifuge
  8. Vortexing
  9. QuantStudio 6 Flex real-time PCR system (Thermo Scientific, USA)
  10. Qubit 2.0 Fluorometer (Life Technologies)
  11. Agilent Bioanalyzer 2100
  12. Illumina Miseq System
  13. Illumina Nextseq 550 System

Software

  1. Quality check of sequence reads: FastQC v0.11.7
    (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
  2. Read mapping: Bowtie2-2.2.9 (Langmead and Salzberg, 2012)
  3. Determination of the normalization factor: deeptools-2.2.4 (Ramirez et al., 2014)
  4. Quantitative analyses: Bwtool (Pohl and Beato, 2014)
  5. Peak calling: homer-v4.8.3 (Heinz et al., 2010)
  6. Peak visualization: IGV-2.3.31 (Robinson et al., 2011)
  7. J-circos-V1 (An et al., 2015)

Procedure

  1. Crosslinking and lysis of mammalian cells and fly tissues
    1. Mouse Neuro-2a cells
      1. Grow mouse Neuro-2a cells to a level of ~106 cells per 10 cm Petri-dish. For this experiment, use ~106 cells. Aspirate and discard the medium. Wash two times each with 4 ml 1x PBS pre-warmed in a 37 °C water bath. Add 1 ml trypsin (Thermo Fisher Scientific, USA) and incubate at 37 °C for 2 min. Quench by adding 3 ml standard cell culture medium. Collect the mixture into a 15 ml centrifuge tube.
      2. Add 108 μl of 37% formaldehyde (Sigma, USA) to a final concentration of 1% (weight/volume) for crosslinking. Rotate at room temperature for 10 min.
      3. Stop crosslinking with 200 μl of 2.5 M glycine (Sigma, USA) for 5 min at room temperature. Centrifuge at 1,000 x g for 3 min at 4 °C. Discard the supernatant.
      4. Resuspend the pellet by adding 1 ml ice-cold 1x PBS with 1x cOmplete proteinase inhibitor (Roche, Germany), and transfer it into a 1.5 ml microcentrifuge tube.
      5. Centrifuge at 1,000 x g for 3 min at 4 °C. After removing the supernatant, wash the pellet two times each with 1 ml ice-cold 1x PBS containing 1x cOmplete proteinase inhibitor (Roche, Germany).
      6. Add 1 ml of 1x RIPA buffer (Sigma, USA) supplemented with 1x cOmplete proteinase inhibitor (Roche, Germany). Incubate at 4 °C for 30 min.
    2. Fly tissues
      For developmental stages, use 1,000 embryos (30 min-1 h after egg-laying), 30 larvae (96 h after egg-laying), and 30 pupae (7 d after egg-laying). For adult stage, use 100 dissected fly muscles and 200 fly heads at each time point.
      1. Collect samples into a 1.5 ml microcentrifuge tube and immediately freeze samples in liquid nitrogen.
      2. Grind tissues into fine powder using mortar and pestle pre-cooled with liquid nitrogen.
      3. Resuspend the fine powder in 1.2 ml 1x PBS. Add 32.4 μl of 37% formaldehyde (Sigma, USA). Incubate at room temperature for 10 min. To quench formaldehyde, add 60 μl of 2.5 M glycine (Sigma, USA). Centrifuge at 5,000 x g for 5 min at 4 °C. After removing the supernatant, wash the pellet three times with 1 ml ice-cold 1x PBS containing 1x cOmplete proteinase inhibitor (Roche, Germany).
      4. Add 1 ml of 1x RIPA buffer (Sigma, USA) supplemented with 1x cOmplete proteinase inhibitor (Roche, Germany). Homogenize the tissue pellet with a glass Dounce tube, and transfer it into a 1.5 ml microcentrifuge tube. Rotate at 4 °C for 1 h.

  2. Sonication
    1. Split the lysate into 250 μl aliquots for four pre-chilled 1.5 ml Bioruptor microtubes (Diagenode, Belgium).
    2. For mouse Neuro-2a cells, sonicate at 4 °C using Bioruptor Pico (Diagenode, Belgium) for 6 cycles with 15 sec on and 15 sec off. For fly tissues, sonicate at 4 °C using Bioruptor Pico (Diagenode, Belgium) for 15 cycles with 30 sec on and 30 sec off.
    3. Transfer sonicated samples into a 1.5 ml microcentrifuge tube.
    4. Centrifuge at 12,000 x g for 20 min at 4 °C, and transfer the supernatant to a new tube.
    5. Store sonicated samples at -80 °C.

  3. Determination of chromatin size and concentration
    1. Use 30 μl aliquot of chromatin sample from the sonicated lysate, and add 90 μl of 1x RIPA buffer (Sigma, USA).
    2. Add 1 μl RNase A (Thermo Scientific, USA), and incubate at 37 °C for 30 min to remove RNA.
    3. To reverse crosslinking, add 5.04 μl of 5 M NaCl (Thermo Scientific, USA), and incubate at 65 °C for 4 h.
    4. Add 1.5 μl of 0.5 M EDTA (Thermo Scientific, USA) and 1.2 μl of 20 mg/μl proteinase K (Sangon Biotech, China) at 55 °C for 2 h.
    5. Isolate DNA by PCR purification kit (QIAGEN, Germany), and elute DNA in 30 μl of Milli-Q water.
    6. Quantify DNA by Qubit dsDNA HS assay kit (Thermo Scientific, USA). A concentration of about 2-3 ng/μl DNA (~30 μl) would be expected from 200 fly heads. 
    7. Examine the chromatin DNA on a 1.5% agarose gel to visualize average size. The optimal size range is between 100 bps and 300 bps. If the chromatin is not in that range, adjust sonication conditions by adding more pulses and repeat Step C3.

  4. Chromatin immunoprecipitation
    1. According to the DNA mass (the volume of fly sample x the concentration of fly sample measured in Procedure C), add 5% (weight/weight) of the mouse epigenome to the fly sample, and mix well. Save 1% (volume/volume) of the sample to a new tube as ChIP input control, and freeze at -20 °C until the elution step.
    2. Add 3 μg antibody, and rotate at 4 °C for 5 h.
    3. Place a magnetic stand (Thermo Scientific, USA) on ice. Add 30 μl Dynabeads (Thermo Scientific, USA) to a 1.5 ml microcentrifuge tube, and wash three times with 1x RIPA buffer (Sigma, USA). Collect beads using magnetic stand (Thermo Scientific, USA), and remove supernatant by aspiration.
    4. Add samples (Fly + Mouse Neuro-2a + antibody) to the pre-washed Dynabeads (Thermo Scientific, USA). Gently mix overnight on a rotator at 4 °C.
    5. Apply ChIP sample to an ice-cold magnetic stand (Thermo Scientific, USA). Remove supernatant by aspiration.
    6. Wash the beads one time with 1x RIPA buffer (Sigma, USA).
    7. Wash the beads two times with ChIP Wash Buffer.
    8. Wash the beads one time with ChIP Final Wash Buffer.

  5. Elution, crosslinking reversal and DNA isolation
    1. Resuspend the beads in 120 μl of ChIP Elution Buffer. Incubate at 65 °C for 30 min.
    2. Add 1 μl RNase A (Thermo Scientific, USA), and incubate at 37 °C for 30 min to remove RNA. At the same time, dilute input sample with 1x RIPA buffer (Sigma, USA) to 120 μl, and incubate with 1 μl RNase A (Thermo Scientific, USA) at 37 °C for 30 min.
    3. To reverse crosslinking, add 5.04 μl of 5 M NaCl (Thermo Scientific, USA), and incubate at 65 °C for 4 h.
    4. Add 1.5 μl of 0.5 M EDTA (Thermo Scientific, USA) and 1.2 μl of 20 mg/μl proteinase K (Sangon Biotech, China) at 55 °C for 2 h.
    5. Isolate DNA by PCR purification kit (QIAGEN, Germany), and elute DNA in 50 μl water.
    6. Quantify DNA by Qubit dsDNA HS assay kit (Thermo Scientific, USA). A concentration of about 0.15-0.3 ng/μl DNA (~50 μl) would be expected from one experiment of immunoprecipitation.

  6. Quality control for ChIP experiment
    1. Performing ChIP-qPCR assays
      1. Design primers to yield PCR product between 100 bps and 200 bps.
      2. Dilute input DNA with RIPA buffer to the same concentration with IP DNA.
      3. Set up real-time PCRs in triplicate with SYBR selected master mix (Thermo Scientific, USA), and the same volume of diluted input DNA and IP DNA.
      4. Mix the samples by vortexing for 2 sec and precipitate samples by brief centrifugation.
      5. Perform real-time PCR with the QuantStudio 6 Flex real-time PCR system (Thermo Scientific, USA) using cycling conditions as shown in the flowing table:


      6. Calculate the fold difference between the experimental sample and negative control.
    2. Preparing high-throughput sequencing library
      1. Use 5-10 ng of DNA harvested by ChIP experiment to generate sequencing library using NEB DNA library prep kit (NEB, USA).
      2. Check the quality of libraries with Bioanalyzer 2100 (Agilent, USA).
      3. Perform quantification by qRT-PCR with a reference to a standard library.
      4. Pool the libraries together in equimolar amounts to a final 2 nM concentration.
      5. Denature the normalized libraries with 0.1 M NaOH (Sigma, USA).
      6. Sequence the pooled libraries on the Miseq/Next-seq platform (Illumina, USA) with single end 100 bps.

Data analysis

The following procedures showed the detailed steps for data analysis (Figure 1).


Figure 1. Overview of ChIP-seq analysis

  1. Sequence quality check
    Use FastQC to assess the read quality by importing data from FastQ files.
  2. Read mapping
    Map Sequence reads to the reference genome dm6 (Drosophila) or mm10 (mouse), respectively with Bowtie2-2.2.9 by default parameters.

    > # Map Sequencing reads to the reference genome mm10
    > nohup bowtie2 -x / seqlib/igenome/Mus_musculus/UCSC/mm10/Sequence/Bowtie2Index/genome -U {sample}.fastq.gz -S {sample}_mm10.sam --no-unal &
    > # Map Sequencing reads to the reference genome dm6
    > nohup bowtie2 -x / seqlib/igenome/Drosophila_melanogaster/UCSC/dm6/Sequence/Bowtie2Index/genome -U {sample}.fastq -S {sample}_dm6.sam --no-unal &
    > # For sample in ChIP_dm6, do the following
    > # Convert file from sam to bam
    > samtools view -Sb {sample}_dm6.sam > {sample}_dm6_nonSorted.bam
    > # Sort BAM file
    > samtools sort {sample}_dm6_nonSorted.bam -o {sample}_dm6_Sorted.bam
    > # Create index files
    > samtools index {sample}_dm6_Sorted.bam

  3. Determination of the normalization factor
    For quantitative comparison, we derive of the scale factor for each sample using the percentage of mapped reads from mouse genome to total reads. Details are as follows:
    1. Combine the number of mapped reads from Drosophila and mouse genomes as total mapped reads for each sample.
      Let:
      α = the spike-in scale factor
      β = the histone modification level
      γ = the percentage of input mouse reads in total input mapped reads
      Nm = the number of mouse mapped reads (in millions) in IP sample
      Nd = the number of Drosophila mapped reads (in millions) in IP sample
    2. Calculate spike-in scale factor as follows.
      α = γ/Nm
    3. Calculate histone modification level as follows.
      β = Nd x α
    4. Normalize the dm6 mapped reads to the scale factor using deeptools-2.2.4 function bamCoverage with 10 bp bin size.

      > # For sample in ChIP_dm6, do the following
      > bamCoverage -b {sample}_ChIP_dm6_Sorted.bam -o {sample}_ChIP_dm6_scaleFactor.bw –scaleFactor α -bs 10 -p 2 –v

  4. Quantitative analysis
    Calculate the ChIP intensity for each gene or region using the Bwtool function bwtool summary with default parameters.

    > bwtool summary {gene}.bed {sample}_ChIP_dm6_scaleFactor.bw {sample}_{gene}_summary.xls –header

  5. Peak calling
    Identify Peak regions by homer-v4.8.3 function findPeaks with parameter "-style histone -F 2 -size 3000 -minDist 5000".

    > makeTagDirectory {sample}_ChIP_tag -fragLength 200 {sample}_ChIP.sam -single
    > makeTagDirectory {sample}_input_tag -fragLength 200 {sample}_input.sam -single
    > findPeaks {sample}_ChIP_tag/ -style histone -o {sample}_size3K_Peaks.xls -i {sample}_input_tag/ -F 2 -size 3000 -minDist 5000 -fragLength 200

  6. Peak visualization
    Display the confident peaks and enriched genome regions by IGV-2.3.31 with the bigwig files generated by bamCoverage.
  7. Peak annotation
    Perform peak annotation using homer function annotatePeaks with a default parameter.

    > annotatePeaks.pl peaks.txt dm6.bed > annotatedPeaks.txt

  8. Functional analyses
    1. Generated scatter plot by R package ggplot2.
      Create a .csv file containing two columns. The first column contains the log2 values of 3 days’ H3K27me3 levels for protein-coding genes. The second column contains the log2 values of 30 days’ H3K27me3 levels for protein-coding genes.
      Generate a scatter plot by R package ggplot2. The results showed that H3K27me3 modification increases with age in head (Figure 2).

      > contour = read.csv(file="all_gene_3_30_log2.csv", header=T)
      > x=contour[,1]
      > y=contour[,2]
      > df = data.frame(x,y)
      > df <- data.frame(x = x, y = y, d = densCols(x, y, colramp = colorRampPalette(rev(rainbow(10,s=1,v=1,start=1/10, end = 7/10,alpha = 1)))))
      > ggplot(df) + geom_point(aes(x, y, col = d), size = 0.01) + geom_density2d(aes(x,y),size=0.3, col="red") + scale_color_identity() + coord_fixed() + geom_abline(slope=1) + scale_y_continuous(limits = c(-4,4)) + scale_x_continuous(limits = c(-4,4))


      Figure 2. Scatter plot showing the comparison result between the H3K27me3 levels of protein-coding genes in 3 d old flies and 30 d ones. Protein-coding genes: the open reading frames of protein-coding genes annotated in dm6. The dm6 mapped reads were normalized to a scale factor to compare the relative H3K27me3 level quantitatively. Each dot on the plot represents a single gene locus. X- and Y-axis represented log2 mean value of gene’s reference-adjusted reads. Contour lines indicated that H3K27me3 signals were higher in aged flies compared to 3 d old flies (Niu and Liu, unpublished).

    2. Generated Circos plot by J-circos-V1 with the bigwig files. The result showed the distribution of H3K27me3 peak profiles (Figure 3).


      Figure 3. Circos plot showing peak profiles of H3K27me3 in 3d and 30d old flies. Black boxes and lines (innermost circle) represented common peak regions, corresponding to their chromosomal locations. Chromosome ideogram was in grey (outermost ring). ChIP-seq was from head tis sues of 3 d and 30 d old male flies. Circos plot of the H3K27me3 epigenome illustrates peak profiles with age in head (Niu and Liu, unpublished).

Recipes

  1. Standard Drosophila media
    Sucrose
    36 g/L
    Maltose
    38 g/L
    Yeast
    22.5 g/L
    Agar
    5.4 g/L
    Maizena
    60 g/L
    Soybean flour
    8.25 g/L
    Sodium benzoate
    0.9 g/L
    Methyl-p-hydroxybenzoate
    0.225 g/L
    Methyl-p-hydroxybenzoate
    6.18 ml/L
    ddH2O to make up 1 L of the food
  2. Glycine, 2.5 M
    1. Weigh 187.6 mg glycine and add 1 ml Milli-Q water
    2. Heat the mixture at 37 °C and mix it until glycine dissolves
    3. Store at room temperature (20-25 °C) for no more than 1 month
  3. ChIP Wash Buffer
    1. Mix 150 mM NaCl, 2 mM EDTA (pH 8.0), 20 mM Tris-HCl (pH 8.0), 1% Triton X-100 (volume/volume), and 0.1% SDS (volume/volume, use 10% SDS solution)
    2. Dilute the solution with Milli-Q water
    3. Store at 4 °C for no more than 6 months
  4. ChIP Final Wash Buffer
    Mix 500 mM NaCl, 2 mM EDTA (pH 8.0), 20 mM Tris-HCl (pH 8.0), 1% Triton X-100 (volume/volume), and 0.1% SDS (volume/volume, use 10% SDS solution).
    Note: Prepare the solution with Milli-Q water. Store at 4 °C for no more than 6 months.
  5. ChIP Elution Buffer
    Mix 100 mM NaHCO3 and 1% SDS (volume/volume, use 10% SDS solution).
    Note: Freshly prepare the solution with Milli-Q water before elution step. Always put the solution at room temperature (20-25 °C) and ensure that there are no SDS precipitates before use.

Acknowledgments

This article is a part of works from our published article (Ma et al., 2018). This work was supported by grants from the National Program on Key Basic Research Project of China to N.L. (2016YFA0501900) and the National Natural Science Foundation of China to N.L. (31371326).

Competing interests

The authors declare that no competing interests exist.

References

  1. An, J., Lai, J., Sajjanhar, A., Batra, J., Wang, C. and Nelson, C. C. (2015). J-Circos: an interactive Circos plotter. Bioinformatics 31(9): 1463-1465.
  2. Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y. C., Laslo, P., Cheng, J. X., Murre, C., Singh, H. and Glass, C. K. (2010). Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell 38(4): 576-589.
  3. Langmead, B. and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4): 357-359.
  4. Ma, Z. J., Wang, H., Cai, Y. P., Wang, H., Niu, K. Y., Wu, X. F., MA, H. H., Yang, Y., Tong, W. H., Liu, F., Liu, Z. D., Zhang, Y. Y., Liu, R., Zhu, Z. J. and Liu, N. (2018). Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila. eLife 7: e35368.
  5. Orlando, D. A., Chen, M. W., Brown, V. E., Solanki, S., Choi, Y. J., Olson, E. R., Fritz, C. C., Bradner, J. E. and Guenther, M. G. (2014). Quantitative ChIP-Seq normalization reveals global modulation of the epigenome. Cell Rep 9(3): 1163-1170.
  6. Pohl, A. and Beato, M. (2014). bwtool: a tool for bigWig files. Bioinformatics 30(11): 1618-1619.
  7. Ramirez, F., Dundar, F., Diehl, S., Gruning, B. A. and Manke, T. (2014). deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res 42(Web Server issue): W187-191.
  8. Robinson, J. T., Thorvaldsdottir, H., Winckler, W., Guttman, M., Lander, E. S., Getz, G. and Mesirov, J. P. (2011). Integrative genomics viewer. Nat Biotechnol 29(1): 24-26.

简介

染色质免疫沉淀,然后测序(ChIP-seq)是实验室中的常规程序; 然而,独立ChIP-seq实验之间的表观基因组范围的定量比较仍然是一个挑战。 在这里,我们提供了一个实验方案,结合计算工作流程,允许使用动物组织定量和比较评估表观基因组。

【背景】 修饰组蛋白的染色质和表观遗传复合物调节DNA对转录机制的可及性,从而允许直接控制基因表达。为了表征组蛋白修饰的表观基因组特征,染色质免疫沉淀然后测序(ChIP-seq)已成为一种广泛使用的方法。然而,传统的ChIP-seq方案本质上不是定量的,因此禁止直接比较源自不同细胞类型的样品或经历过不同遗传或化学扰动的细胞。尽管已经提出了几种 in silico 归一化方法来克服这个缺点,但仍然缺乏基于实验的策略。 2014年,奥兰多等人(2014)开发了一种名为ChIP的方法,该方法使用参考外源基因组(ChIP-Rx),该方法利用恒定量的参考或''spike-in''表观基因组基于细胞的表观基因组比较。在当前的协议中,我们通过使用映射的尖峰参考表观基因组的百分比来改进该方法。我们已成功应用此协议直接比较来自动物组织的两个或更多ChIP-seq数据集。

关键字:表观基因组, H3K27me3, 定量 ChIP-seq, 外源参照, 果蝇

材料和试剂

  1. 耗材
    1. 移液器吸头
    2. 1.5毫升微量离心管
    3. 15毫升微量离心管
    4. 1.5 ml Bioruptor Microtubes(Diagenode,目录号:C30010016)
    5. 10厘米培养皿
    6. 玻璃弹跳管

  2. 生物材料
    1. 小鼠Neuro-2a细胞
    2. Drosophila (1000个胚胎[产蛋后30分钟 - 1小时],30个幼虫[产卵后96小时]和30个蛹[产蛋后7天])
      起始材料:细胞在含有10%FBS的完全LG-DMEM(Thermo Fisher Scientific,Life Technologies,目录号:11995-065)中于37℃,5%CO 2 和饱和湿度下培养。 (Sigma-Aldrich,目录号:12003C),在10cm培养皿中以1.0×10 6个细胞/ cm 2 接种。除非另有说明,否则将蝇在标准的 Drosophila 培养基(配方1)中在25℃,60%湿度,12小时光照和12小时黑暗循环中培养。

  3. 试剂
    ChIP试剂
    1. 甲醛,37%(重量/体积)(Sigma-Aldrich,目录号:252549),在室温(20-25°C)下储存
    2. 甘氨酸(Sigma-Aldrich,目录号:G7403),在室温(20-25°C)下保存
    3. PBS缓冲液,20x(Sangon Biotech,目录号:B548117),在室温(20-25°C)下储存
    4. RIPA缓冲液(Sigma-Aldrich,目录号:R0278),储存在4℃
    5. Tris buffer,1 M,PH 8.0(Sangon Biotech,目录号:B548127),在室温(20-25°C)下保存
    6. 氯化钠,5 M(Thermo Fisher Scientific,Invitrogen TM ,目录号:AM9759),在室温(20-25°C)下储存
    7. Triton X-100(Sigma-Aldrich,目录号:T8787),储存在4℃
    8. EDTA,0.5 M,pH 8.0(Thermo Fisher Scientific,Invitrogen TM ,目录号:AM9260G),在室温(20-25°C)下储存
    9. SDS,10%(重量/体积)(Sangon Biotech,目录号:B548118),在室温(20-25°C)下储存
    10. c完整蛋白酶抑制剂鸡尾酒片(Roche Diagnostics,目录号:11697498001),储存于4°C
    11. Dynabeads蛋白G(Thermo Fisher Scientific,Invitrogen TM ,目录号:10004D),储存于4°C
    12. 抗三甲基组蛋白H3(Lys27)抗体(Merck,目录号:07-449),在-20°C保存
    13. 碳酸氢钠(Sangon Biotech,目录号:A100865),在室温(20-25°C)下储存
    14. 蛋白酶K(Sangon Biotech,目录号:A600451),储存在-20℃
    15. RNase A,10 mg / ml(Thermo Fisher Scientific,目录号:EN0531),储存于-20°C
    16. PCR纯化试剂盒(QIAGEN,目录号:28106),在室温(20-25°C)下保存
    17. Qubit dsDNA HS检测试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q32854),储存于4°C(警告:避光)
    18. 胰蛋白酶(Thermo Fisher Scientific,目录号:25300054)
    19. 液氮
    20. 甘氨酸,2.5米(见食谱)
    21. ChIP洗涤缓冲液(见食谱)
    22. ChIP最终洗涤缓冲液(参见食谱)
    23. ChIP洗脱缓冲液(见食谱)

    库试剂
    1. Agencourt AMPure XP(SPRI珠; Beckman Coulter,目录号:A63881),储存于4°C
    2. 用于Illumina的NEBNext Ultra DNA文库制备试剂盒(New England Biolabs,目录号:E7370L),储存于-20°C
    3. 用于Illumina的NEBNext Multiplex Oligos(New England Biolabs,目录号:E7335L),储存于-20°C
    4. 凝胶提取试剂盒(QIAGEN,目录号:20021),在室温(20-25°C)下保存
    5. 氢氧化钠溶液,10 M(Sigma-Aldrich,目录号:72068)

设备

  1. Bioruptor Pico(Diagenode)
  2. DynaMag-2磁铁(赛默飞世尔科技,目录号:12321D)
  3. 研钵和研杵
  4. 1毫升玻璃均化器
  5. 水浴
  6. 旋转
  7. 离心分离机
  8. 涡旋
  9. QuantStudio 6 Flex实时PCR系统(Thermo Scientific,USA)
  10. Qubit 2.0荧光计(Life Technologies)
  11. Agilent Bioanalyzer 2100
  12. Illumina Miseq系统
  13. Illumina Nextseq 550系统

软件

  1. 序列的质量检查读取:FastQC v0.11.7
    (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
  2. 阅读映射:Bowtie2-2.2.9(Langmead and Salzberg,2012)
  3. 标准化因子的确定:deeptools-2.2.4(Ramirez et al。,2014)
  4. 定量分析:Bwtool(Pohl和Beato,2014)
  5. 高峰召唤:本垒打-V4.8.3(Heinz et al。,2010)
  6. 峰值可视化:IGV-2.3.31(Robinson et al。,2011)
  7. J-circos-V1(An et al。,2015)

程序

  1. 哺乳动物细胞和苍蝇组织的交联和裂解
    1. 小鼠Neuro-2a细胞
      1. 将小鼠Neuro-2a细胞培养至每10cm培养皿~10 6 细胞的水平。对于该实验,使用~10 6 细胞。吸出并丢弃介质。用在37℃水浴中预热的4ml 1x PBS洗涤两次。加入1ml胰蛋白酶(Thermo Fisher Scientific,USA)并在37℃下孵育2分钟。加入3ml标准细胞培养基淬灭。将混合物收集到15ml离心管中。
      2. 加入108μl37%甲醛(Sigma,USA)至终浓度为1%(重量/体积)进行交联。在室温下旋转10分钟。
      3. 在室温下用200μl2.5M甘氨酸(Sigma,USA)停止交联5分钟。在4℃下以1,000 x g 离心3分钟。丢弃上清液。
      4. 通过加入1ml冰冷的1x PBS和1x cOmplete蛋白酶抑制剂(Roche,Germany)重悬沉淀,并将其转移到1.5ml微量离心管中。
      5. 在4℃下以1,000 x g 离心3分钟。除去上清液后,用含有1x完全蛋白酶抑制剂(Roche,Germany)的1ml冰冷的1x PBS洗涤沉淀两次。
      6. 加入1ml补充有1x cOmplete蛋白酶抑制剂(Roche,Germany)的1x RIPA缓冲液(Sigma,USA)。在4°C孵育30分钟。
    2. 飞组织
      对于发育阶段,使用1,000个胚胎(产卵后30分钟-1小时),30个幼虫(产卵后96小时)和30个蛹(产卵后7天)。对于成人阶段,在每个时间点使用100个解剖的飞肌和200个飞行头。
      1. 将样品收集到1.5ml微量离心管中并立即在液氮中冷冻样品。
      2. 使用用液氮预冷却的研钵和研杵将组织研磨成细粉。
      3. 将细粉末重悬于1.2ml 1x PBS中。加入32.4μl37%甲醛(Sigma,USA)。在室温下孵育10分钟。为了淬灭甲醛,加入60μl2.5M甘氨酸(Sigma,USA)。在4℃下以5,000 x g 离心5分钟。除去上清液后,用含有1x cOmplete蛋白酶抑制剂(Roche,Germany)的1ml冰冷的1x PBS洗涤沉淀三次。
      4. 加入1ml补充有1x cOmplete蛋白酶抑制剂(Roche,Germany)的1x RIPA缓冲液(Sigma,USA)。用玻璃Dounce管将组织颗粒均化,并将其转移到1.5ml微量离心管中。在4°C旋转1小时。

  2. 超声化
    1. 将裂解物分成250μl等分试样,用于四个预冷的1.5ml Bioruptor微管(Diagenode,Belgium)。
    2. 对于小鼠Neuro-2a细胞,使用Bioruptor Pico(比利时Diagenode)在4℃下超声处理6个循环,15秒开启和15秒关闭。对于苍蝇组织,使用Bioruptor Pico(比利时Diagenode)在4℃下超声处理15个循环,30秒开启和30秒关闭。
    3. 将超声处理的样品转移到1.5ml微量离心管中。
    4. 在4℃下以12,000 x g 离心20分钟,并将上清液转移到新管中。
    5. 将超声处理的样品保存在-80°C。

  3. 染色质大小和浓度的测定
    1. 使用来自超声裂解物的30μl等份的染色质样品,并添加90μl的1x RIPA缓冲液(Sigma,USA)。
    2. 加入1μlRNaseA(Thermo Scientific,USA),在37°C孵育30分钟以除去RNA。
    3. 为了逆转交联,加入5.04μl的5M NaCl(Thermo Scientific,USA),并在65℃下孵育4小时。
    4. 在55℃下加入1.5μl0.5MEDTA(Thermo Scientific,USA)和1.2μl20mg/μl蛋白酶K(Sangon Biotech,China)2小时。
    5. 通过PCR纯化试剂盒(QIAGEN,Germany)分离DNA,并在30μlMilli-Q水中洗脱DNA。
    6. 通过Qubit dsDNA HS测定试剂盒(Thermo Scientific,USA)定量DNA。 200个飞行头的浓度约为2-3 ng /μlDNA(~30μl)。&nbsp;
    7. 检查1.5%琼脂糖凝胶上的染色质DNA,以显示平均大小。最佳尺寸范围介于100 bps和300 bps之间。如果染色质不在该范围内,请通过添加更多脉冲来调整超声处理条件并重复步骤C3。

  4. 染色质免疫沉淀
    1. 根据DNA质量(蝇样品的体积×在方法C中测量的蝇样品的浓度),将5%(重量/重量)的小鼠表观基因组加入到蝇样品中,并充分混合。将1%(体积/体积)样品保存到新管中作为ChIP输入对照,并在-20℃下冷冻直至洗脱步骤。
    2. 加入3μg抗体,在4°C旋转5小时。
    3. 将磁力架(Thermo Scientific,USA)放在冰上。将30μlDynababads(Thermo Scientific,USA)加入1.5ml微量离心管中,并用1x RIPA缓冲液(Sigma,USA)洗涤三次。使用磁力架(Thermo Scientific,USA)收集珠子,并通过抽吸除去上清液。
    4. 将样品(Fly + Mouse Neuro-2a +抗体)加入预洗涤的Dynabeads(Thermo Scientific,USA)中。在4°C的旋转器上轻轻混合过夜。
    5. 将ChIP样品应用于冰冷的磁力架(Thermo Scientific,USA)。通过抽吸除去上清液。
    6. 用1x RIPA缓冲液(Sigma,USA)洗涤珠子一次。
    7. 用ChIP洗涤缓冲液洗涤珠子两次。
    8. 用ChIP最终洗涤缓冲液洗涤珠子一次。

  5. 洗脱,交联逆转和DNA分离
    1. 将珠子重悬于120μlChIP洗脱缓冲液中。在65°C孵育30分钟。
    2. 加入1μlRNaseA(Thermo Scientific,USA),在37°C孵育30分钟以除去RNA。同时,用1x RIPA缓冲液(Sigma,USA)将输入样品稀释至120μl,并与1μlRNaseA(Thermo Scientific,USA)在37℃下孵育30分钟。
    3. 为了逆转交联,加入5.04μl的5M NaCl(Thermo Scientific,USA),并在65℃下孵育4小时。
    4. 在55℃下加入1.5μl0.5MEDTA(Thermo Scientific,USA)和1.2μl20mg/μl蛋白酶K(Sangon Biotech,China)2小时。
    5. 通过PCR纯化试剂盒(QIAGEN,Germany)分离DNA,并在50μl水中洗脱DNA。
    6. 通过Qubit dsDNA HS测定试剂盒(Thermo Scientific,USA)定量DNA。从一次免疫沉淀实验可以预期浓度为约0.15-0.3ng /μlDNA(~50μl)。

  6. ChIP实验的质量控制
    1. 进行ChIP-qPCR检测
      1. 设计引物以产生100 bps和200 bps之间的PCR产物。
      2. 用RIPA缓冲液将输入DNA稀释至与IP DNA相同的浓度。
      3. 使用SYBR选择的主混合物(Thermo Scientific,USA)和相同体积的稀释输入DNA和IP DNA一式三份设置实时PCR。
      4. 通过涡旋混合样品2秒并通过短暂离心沉淀样品。
      5. 使用QuantStudio 6 Flex实时PCR系统(Thermo Scientific,USA)使用循环条件进行实时PCR,如流程表所示:


      6. 计算实验样品和阴性对照之间的倍数差异。
    2. 准备高通量测序库
      1. 使用通过ChIP实验收获的5-10ng DNA,使用NEB DNA文库制备试剂盒(NEB,USA)产生测序文库。
      2. 使用Bioanalyzer 2100(Agilent,USA)检查文库的质量。
      3. 通过参考标准库的qRT-PCR进行定量。
      4. 将文库以等摩尔量汇集到最终的2nM浓度。
      5. 用0.1M NaOH(Sigma,USA)使标准化文库变性。
      6. 在Miseq / Next-seq平台(Illumina,USA)上对合并的文库进行测序,单端100 bps。

数据分析

以下程序显示了数据分析的详细步骤(图1)。


图1. ChIP-seq分析概述

  1. 序列质量检查
    使用FastQC通过从FastQ文件导入数据来评估读取质量。
  2. 阅读映射
    Map序列使用Bowtie2-2.2.9默认参数分别读取参考基因组dm6( Drosophila )或mm10(小鼠)。

    &GT; #Map测序读取到参考基因组mm10
    &GT; nohup bowtie2 -x / seqlib / igenome / Mus_musculus / UCSC / mm10 / Sequence / Bowtie2Index / genome -U {sample} .fastq.gz -S {sample} _mm10.sam --no-unal&amp;
    &GT; #Map测序读取到参考基因组dm6
    &GT; nohup bowtie2 -x / seqlib / igenome / Drosophila_melanogaster / UCSC / dm6 / Sequence / Bowtie2Index / genome -U {sample} .fastq -S {sample} _dm6.sam --no-unal&amp;
    &GT; #对于ChIP_dm6中的示例,请执行以下操作
    &GT; #将文件从山姆转换为bam
    &GT; samtools view -Sb {sample} _dm6.sam&gt; {sample} _dm6_nonSorted.bam
    &GT; #排序BAM文件
    &GT; samtools sort {sample} _dm6_nonSorted.bam -o {sample} _dm6_Sorted.bam
    &GT; #创建索引文件
    &GT; samtools index {sample} _dm6_Sorted.bam

  3. 确定归一化因子
    对于定量比较,我们使用从小鼠基因组到总读数的映射读数的百分比得出每个样品的比例因子。详情如下:
    1. 将来自 Drosophila 和小鼠基因组的映射读数的数量合并为每个样本的总映射读数。
      让我们:
      α=加标比例因子
      β=组蛋白修饰水平
      γ=总输入映射读数中输入鼠标读数的百分比
      Nm = IP样本中鼠标映射读数的数量(以百万计)
      Nd = IP样本中 Drosophila 映射读数(以百万计)的数量
    2. 计算加标比例因子如下。
      α=γ/ Nm
    3. 计算组蛋白修饰水平如下。
      β= Nd×α
    4. 使用具有10 bp bin大小的deeptools-2.2.4函数bamCoverage将dm6映射读取标准化为比例因子。

      &GT; #对于ChIP_dm6中的示例,请执行以下操作
      &GT; bamCoverage -b {sample} _ChIP_dm6_Sorted.bam -o {sample} _ChIP_dm6_scaleFactor.bw-scaleFactorα-bs 10 -p 2 -v

  4. 定量分析
    使用Bwtool函数bwtool摘要和默认参数计算每个基因或区域的ChIP强度。

    &GT; bwtool summary {gene} .bed {sample} _ChIP_dm6_scaleFactor.bw {sample} _ {gene} _summary.xls -header

  5. 高峰呼唤
    通过homer-v4.8.3函数findPeaks识别峰值区域,参数为“-style组蛋白-F 2 -size 3000 -minDist 5000”。

    &GT; makeTagDirectory {sample} _ChIP_tag -fragLength 200 {sample} _ChIP.sam -single
    &GT; makeTagDirectory {sample} _input_tag -fragLength 200 {sample} _input.sam -single
    &GT; findPeaks {sample} _ChIP_tag / -style histone -o {sample} _size3K_Peaks.xls -i {sample} _input_tag / -F 2 -size 3000 -minDist 5000 -fragLength 200

  6. 峰值可视化
    使用bamCoverage生成的bigwig文件,通过IGV-2.3.31显示自信峰和丰富的基因组区域。
  7. 峰值注释
    使用带有默认参数的本垒打函数 annotatePeaks 执行峰值注释。

    &GT; annotatePeaks.pl peaks.txt dm6.bed&gt; annotatedPeaks.txt

  8. 功能分析
    1. 由R package ggplot2生成的散点图。
      创建包含两列的.csv文件。第一列包含蛋白质编码基因的3天H3K27me3水平的log2值。第二列包含蛋白质编码基因的30天H3K27me3水平的log2值。
      通过R package ggplot2生成散点图。结果显示H3K27me3修饰随着头部年龄的增加而增加(图2)。

      &GT; contour = read.csv(file =“all_gene_3_30_log2.csv”,header = T)
      &GT; x =轮廓[,1]
      &GT; y =轮廓[,2]
      &GT; df = data.frame(x,y)
      &GT; df&lt; - data.frame(x = x,y = y,d = densCols(x,y,colramp = colorRampPalette(rev(rainbow(10,s = 1,v = 1,start = 1/10,end = 7/10,alpha = 1)))))
      &GT; ggplot(df)+ geom_point(aes(x,y,col = d),size = 0.01)+ geom_density2d(aes(x,y),size = 0.3,col =“red”)+ scale_color_identity()+ coord_fixed() + geom_abline(slope = 1)+ scale_y_continuous(limits = c(-4,4))+ scale_x_continuous(limits = c(-4,4))


      图2.散点图显示3 d蝇和30 d中蛋白质编码基因的H3K27me3水平之间的比较结果。蛋白质编码基因:蛋白质编码基因的开放阅读框架注释在dm6。将dm6定位的读数标准化为比例因子以定量比较相对H3K27me3水平。图中的每个点代表单个基因座。 X轴和Y轴代表基因参考调整读数的log2平均值。等高线表明老龄果蝇中H3K27me3信号高于3龄蝇(Niu和Liu,未发表)。

    2. 由J-circos-V1和bigwig文件生成的Circos图。结果显示H3K27me3峰分布的分布(图3)。


      图3. Circos图显示了3d和30d老蝇中H3K27me3的峰轮廓。黑框和线(最内圈)代表共同的峰区,对应于它们的染色体位置。染色体表意文字为灰色(最外环)。 ChIP-seq来自3 d和30 d雄性苍蝇的头部起诉。 H3K27me3表观基因组的Circos图显示了头部年龄的峰值谱(Niu和Liu,未发表)。

食谱

  1. 标准 Drosophila 媒体
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>蔗糖
    36克/升
    麦芽糖
    38克/升
    酵母
    22.5克/升
    琼脂
    5.4克/升
    Maizena
    60克/升
    大豆粉
    8.25克/升
    苯甲酸钠
    0.9克/升
    对羟基苯甲酸甲酯
    0.225克/升
    对羟基苯甲酸甲酯
    6.18 ml / L
    ddH 2 O以补充1升食物
  2. 甘氨酸,2.5 M
    1. 称取187.6mg甘氨酸并加入1ml Milli-Q水
    2. 将混合物在37℃加热并混合直至甘氨酸溶解
    3. 在室温(20-25°C)下储存不超过1个月
  3. ChIP洗涤缓冲液
    1. 混合150 mM NaCl,2 mM EDTA(pH 8.0),20 mM Tris-HCl(pH 8.0),1%Triton X-100(体积/体积)和0.1%SDS(体积/体积,使用10%SDS溶液)
    2. 用Milli-Q水稀释溶液
    3. 在4°C下储存不超过6个月
  4. ChIP最终洗涤缓冲液
    混合500 mM NaCl,2 mM EDTA(pH 8.0),20 mM Tris-HCl(pH 8.0),1%Triton X-100(体积/体积)和0.1%SDS(体积/体积,使用10%SDS溶液) 。
    注意:用Milli-Q水制备溶液。在4°C下储存不超过6个月。
  5. ChIP洗脱缓冲液
    混合100 mM NaHCO 3 和1%SDS(体积/体积,使用10%SDS溶液)。
    注意:在洗脱步骤前用Milli-Q水新鲜制备溶液。始终将溶液置于室温(20-25°C)并确保在使用前没有SDS沉淀。

致谢

本文是我们发表的文章(Ma et al。,2018)的作品的一部分。这项工作得到了中国国家重点基础研究项目国家计划的资助。 (2016YFA0501900)和中国国家自然科学基金会(31371326)。作者声明不存在竞争利益。

参考

  1. An,J.,Lai,J.,Sajjanhar,A.,Batra,J.,Wang,C。和Nelson,C.C。(2015)。 J-Circos:交互式Circos绘图仪。 生物信息学 31(9):1463-1465。
  2. Heinz,S.,Benner,C.,Spann,N.,Bertolino,E.,Lin,Y.C.,Laslo,P.,Cheng,J.X.,Murre,C.,Singh,H。and Glass,C.K。(2010)。 谱系决定转录因子的简单组合是巨噬细胞和B细胞身份所需的顺式调节元件。 Mol Cell 38(4):576-589。
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Copyright Niu et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Niu, K., Liu, R. and Liu, N. (2018). Quantitative ChIP-seq by Adding Spike-in from Another Species. Bio-protocol 8(16): e2981. DOI: 10.21769/BioProtoc.2981.
  2. Ma, Z. J., Wang, H., Cai, Y. P., Wang, H., Niu, K. Y., Wu, X. F., MA, H. H., Yang, Y., Tong, W. H., Liu, F., Liu, Z. D., Zhang, Y. Y., Liu, R., Zhu, Z. J. and Liu, N. (2018). Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila. eLife 7: e35368.
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