Chromatin Immunoprecipitation, sequencing and data analysis in Drosophila S2 cells

Chromatin Immunoprecipitation, sequencing and data analysis in Drosophila S2 cells

Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) is a technique to determine the binding pattern of a protein with different regions of chromatin or with specific nascent mRNAs. The ChIP-Seq involves fixation, chromatin fragmentation, immunoprecipitation, DNA purification and analysis. The first step of ChIP is the fixation of living cells that crosslinks the protein with chromatin and then the fragmentation of that chromatin by physical shearing or DNA enzymatic digestion. Immunoprecipitation isolates the fragments of chromatin-associated with a particular protein and then purification involves removal of chromatin-associated protein and RNA to obtain purified DNA fragments. Those purified DNA fragments are analysed initially by quantitative real-time PCR and then used for high throughput sequencing to determine the genome wide binding pattern of our protein of interest.

Chromatin Immunoprecipitation and sequencing protocol

        A. Fixation

1. Grow the Drosophila S2 cell line in a T75 flask having Insect-XPRESS protein-free insect cell medium (BELN12-730Q) till their growth reaches 90% confluence.
2. Harvest the cells by tapping the flask with the hand or by a forced flow of media on cells with a pipette. Count the number of cells with a hemocytometer and take 4 × 10⁷ cells for each set of ChIP experiment.
   Note: The S2 cells are partially adherent to the plastic surface and therefore they get easily detached by gentling tapping on the flask. The lower surface of the flask with confluent cells looks opaque from the outside and will become transparent as the cells detached from the surface. 
3. Resuspend the cells in 10 mL 1XPBS and centrifuge at 1000 g for 5 min. Repeat the step to wash the cells twice.
4. Incubate the cells with 1% formaldehyde (Polysciences, 04018-1) for 10 min at RT on a gently revolving rotator flatform to fix them. 
5. Incubate the cells with 125 mM glycine on the rotator for 5 min at RT to stop the fixation process. 
   Note: Proper fixation is a crucial step in the ChIP experiment as under or over-fixation of cells have a large change in the final result.
   
   B. Chromatin Fragmentation:
    
6. Wash the cells twice with ice-cold 1X PBS and store the cell pellet at -80⁰C. 
   Note: Although these cells could be stored for up to 6 months, we always processed them straight away and never stored them at -80⁰C.
7. Resuspend the cell pellet in 1 mL cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) supplemented with 1X cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, 5056489001) and 1X PhosStop (Sigma-Aldrich, 4906837001) and incubate them for 10 min at 4°C on ice with intermittent mixing.
   Note: Treatment with cell lysis buffer solubilise the cell membrane so that the cytoplasmic content leaks out in the buffer.
8. Remove the cell lysis buffer by centrifugation and resuspend the cell pellet in 1 mL nuclear lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1.0% SDS) supplemented with 1X cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail and 1X PhosStop and incubate them for 10 min at 4°C on ice with intermittent mixing. 
9. Add additional 500 µL IP dilution buffer (16.7 mM Tris pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS), mix them by pipetting and aliquot in 300 µL volume in 1.5 mL Bioruptor Plus TPX microtubes (Diagenode, C30010010-300).
10. Place the tubes with samples in the adaptor and put the adaptor in Bioruptor Sonicator (Diagenode). Sonicate the cells for 5 cycles of 30 second ON - 30 second OFF at 4⁰C with maximum power intensity. 
    Note: The sonication parameters depend on the individual sonicator, plastic tubes used and varies with cell types and culture conditions and it needs to be standardized, as follows, for different types of cells and experimental conditions. 
    
    C. Immunoprecipitation
11. Centrifuge the sonicated sample at 13000 RPM for 20 min and transfer all clear supernatant samples to a  fresh 15 mL tube. 
12. Take an aliquot of 100 µL supernatant in a 1.5 mL tube for DNA purification, which will be used as input sample and determination of average fragment size. 
13. Extract the DNA (as described later) and run a small fraction in agarose gel to examine the fragment size which should be approximately 500 + 300 bp. If the fragment size is in this range, proceed to next step otherwise repeat the experiment with additional or fewer sonication cycles if the fragment size is bigger or smaller, respectively.
14. Dilute the remaining 1.4 mL cleared supernatant with 7.0 mL (1:5) IP dilution buffer in a 15 mL tube at 4⁰C on ice. For each ChIP, typically add 5 to 10 µg of antibody to the supernatant and incubate overnight at 4°C on a rotator. This will make the antibody-protein-DNA complex in the solution.  Here we have used 5 µg of Pol II antibody for each ChIP. 
    Note: The antibody can alternatively be attached to magnetic beads (see below) prior to adding the beads to the chromatin.
15. Add prewashed 20 µL Dynabeads Protein G or Protein A to the lysate-antibody mix and incubated them further for 2 hrs at 4°C on a rotator. 
    Note: Protein G and Protein A are bacterial proteins that have a strong affinity for the Fc region of polyclonal or monoclonal IgG-type antibodies. Both these proteins have a variable binding affinity with different immunoglobulins and therefore antibodies raised in different organisms or different classes of immunoglobulins have a variable affinity with these two proteins.
16. Wash beads 6 times with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris pH 8.0, 500 mM NaCl) and once with 1X TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) using a magnetic stand. 
17. Incubate beads with 250 µL elution buffer (0.1M NaHC03, 1% SDS) on a rotator for 15 min at RT and collect the clear supernatant by putting the tube on the magnetic stand for 5 min.
    
    D. DNA purification and 
     
18. Reverse cross-link of the eluted chromatin by adding 38 µL de-crosslinking buffer (2M NaCl, 0.1M EDTA, 0.4M Tris pH 7.5) and then incubate them on rotator at 65°C overnight. 
19. Add 2 µL Proteinase K (50 mg/mL) and incubate on a rotator at 50°C for 2 hrs to digest the protein. 
20. Purify the DNA from ChIP and Input samples by using Monarch PCR and DNA Cleanup Kit (New England Biolab, T1030S) and quantitate the amount of DNA using Qubit™ dsDNA HS Assay Kit (Thermofisher Scientific, Q32851).
21. Quantify the efficiency of ChIP by Real-Time qPCR with the SensiFAST SYBR Hi-ROX Kit (Bioline, BIO-92005) using an ABI PRISM 7000 Real-Time PCR system (Applied Biosystems). 
22. Perform the qPCR by using primers from the genomic region of interest along with the positive and negative controls and quantify the fold enrichment and percentage input of ChIP DNA by using input DNA as a loading control.
23. Following the confirmation of positive ChIP signal by qPCR, a library was constructed and sequenced as described below.
24. To prepare the library for NGS sequencing, sonicate the ChIP and input DNA samples further to 200 bp fragment size by using a Bioruptor Pico (Diagenode). 
25. Make the ChIP-DNA library by using the NEBNext Ultra II DNA Library Prep Kit (New England Biolab, E7645L) and NEBnext Multiplex Oligos for Illumina Dual Index Primers (New England Biolabs, E7600S), using 10 ng of fragmented ChIP DNA and the kits recommended protocols. 
26. Assess the quality of constructed libraries by using the Tapestation 2200 (Agilent G2964AA) with High Sensitivity D1000 DNA ScreenTape (Agilent 5067–5584). Libraries were tagged with unique barcodes and sequenced simultaneously on a HiSeq 4000 sequencing system.
    
    E. Data Analysis
     
27. ChIP-Seq data can be visualized and also analyzed by using the user-friendly bioinformatics software package Lasergene Genomics Suite Version 14 (DNASTAR). 
28. Pre-process, assemble and map the sequencing reads in the FASTQ files of ChIP-Seq data by using the SeqMan NGen software of this package after selecting the NCBI D. melanogaster Dm6 genome release and accompanying annotations.
29. Analyse the assembly and alignment output files for each genome contig with the ArrayStar and Gen-Vision Pro software (from the same package). Save the profile from a selected region as high-resolution images. 
30. Download an index for Dm6 from the HISAT2 website to perform the metagene analysis.
31. Align the FASTQ files by using HISAT2 v2.1.0.
32. Convert the resulting SAM files to BAM format and then sort and index them with Samtools 1.6.
33. Convert the BAM files into Bedgraph files with the genomeCoverageBed command and options -bga and -ibam from the Bedtools v2.26.0 suite.
34. Use the Custom Perl scripts to filter the Dm6 annotations for genes separated by a minimum distance to avoid overlapping signals.
35. Subsequently, use the custom scripts to extract the signal from the Bedgraph files for each entry in the filtered gene list. 
36. Use a single base resolution for flanking regions, while bin the signal in gene bodies into 16 bins to take account of different gene lengths. 
37. Normalize each dataset by the total mapped sequencing reads in that dataset.
38. Cross-reference between different datasets based on the ‘name’ field, after filtering the annotations for multiple entries with the identical name. 
39. Use a custom script to extract the sequencing read coverage from the Bedgraph files for each exon/intron/exon region in the dm6 annotation file (downloaded from UCSC Table Browser) into the file xon_fly_gene.
40. To analyse the protein enrichment at exon to intron in the gene body, divide the signal of each exon or intron by the average coverage in the input sample to normalise for any bias in the sequencing.
41. Compare the fold change of ChIP signal/Input signal in intron to that of their flanking exons using Wilcox.test (two-sided and unpaired) in R (www.r-project.org). Analysis of data can be done with a given range of intron length.
42. Analysis with the longer introns is considered to be more informative because of the predicted lower resolution of ChIP at discriminating between closely adjacent sequences and because of the lower sequencing coverage of shorter introns compared to longer introns. 
43. Deposit all ChIP-seq raw sequencing data and Bedgraph files in the GEO repository. 

Note: All custom scripts used in this study are provided in Source Code File 1 in Singh et al, 2019.