ChIP-seq analysis in Neurospora crassa

Summary/Abstract

Chromatin immunoprecipitation paired with next generation sequencing (ChIP-seq) can be used to determine genome-wide distribution of transcriptions factors, transcriptional machinery, or histone modifications. DNA-protein interactions are covalently crosslinked with the addition of formaldehyde. Chromatin is prepared and sheared, then immunoprecipitated with the appropriate antibody. After reversal of crosslinking and treating with protease, the resulting DNA fragments are sequenced and mapped to the reference genome to determine overall enrichment. Here we describe a method of ChIP-seq for investigating protein-DNA interactions in the filamentous fungus Neurospora crassa.

1. Introduction

Protein-DNA interactions regulate diverse nuclear processes such as gene expression, DNA repair and maintenance, chromosome segregation, and establishing and maintaining epigenetic modifications. The advent of chromatin immunoprecipitation (ChIP) has proven to be critical in the study of protein-DNA interactions and associated processes. ChIP followed by high throughput sequencing (ChIP-seq) has become a standard method in genome biology, allowing researchers to look at diverse DNA-protein interactions, including histone occupation, transcription factor binding, histone modifications, histone turnover, and other features of the genome-wide chromatin landscape including base modifications.

ChIP was first described by Gilmour and Lis [1] as a method to investigate localization of regulatory factors such as RNA polymerase II (Pol II) and histone occupation in Drosophila [2]. These original studies were performed with UV crosslinking followed by restriction digest and Southern blotting. Reversible formaldehyde crosslinking was introduced by Solomon et al. [3] to determine the association of Pol II with heat shock protein (hsp) genes in Drosophila. Chromatin was fragmented via sonication or restriction digest followed by immunoprecipitation of covalently crosslinked protein-DNA complexes with the appropriate antibodies. After immunoprecipitation, crosslinking of immunoprecipitated protein-DNA complexes was reversed with heat, and remaining DNA fragments were analyzed by Southern blot. Reversible crosslinking has allowed for the advancement of ChIP applications, including ChIP followed by microarray (ChIP-chip), ChIP followed by quantitative polymerase chain reaction (ChIP-qPCR), and ChIP followed by next generation sequencing (ChIP-seq).

Early application of ChIP in fungi was described in Saccharomyces cerevisiae [4] and Schizosaccharomyces pombe [5]. Here we describe a ChIP-seq method for use in the filamentous fungus Neurospora crassa. which is a derivation of the protocol originally developed by Tamaru and colleagues [6]. Chromatin fractions are prepared by covalent formaldehyde crosslinking and fragmentation by sonication. Fragmented chromatin is then immunoprecipitated with the appropriate antibody bound to agarose beads. Covalent crosslinking of protein-DNA complexes is then reversed by heat, and the chromatin fractions are treated with RNase and proteinase. Remaining DNA fragments are purified, and Illumina sequencing libraries are then prepared and sequenced (Figure 1). Additionally, we provide a brief summary of available methods for downstream analysis of sequencing results and a sample pipeline for data analysis (Figure 2).

Figure 1. Schematic diagram of a ChIP-seq experiment.

DNA-binding proteins are covalently cross-linked to chromatin in vivo. The chromatin fiber is sheared by sonication into small fragments, which are subjected to immunoprecipitation using an antibody that binds a specific DNA-binding protein. Shown here as a transcription factor (TF). Following immunoprecipitation, the crosslinks are reversed and DNA is purified, sequenced, and analyzed.

Figure 2. General bioinformatics workflow for ChIP-seq analysis.

Individual ChIP-seq analysis pipelines will vary based on the specific ChIP-seq application, but analyses workflows include several basic steps. Most ChIP-seq experiments in fungi will require a minimum of 1 – 4 million sequence reads generated using an Illumina sequencing instrument. Raw sequence reads should be pre-processed using a program such as FastQC [7], to remove Illumina adaptor sequences, and remove PCR and optical duplicates. Pre-processed reads are then aligned to a reference genome using a short read aligner such as bowtie2 [8] or the Burrows-Wheeler Aligner [9]. Aligned reads can be visualized using genome browser software, such as the Broad Integrative Genomics Viewer [10] or Gbrowse [11]. Aligned reads can then analyzed using a variety of software packages, depending on the specific goals of the ChIP-seq. For example, software such as HOMER [12], MACS [13], or SICER [14] can be used to call peaks, identify DNA sequence motifs, or perform differential enrichment analyses.

2. Materials

2.1. Chromatin Immunoprecipitation:

1. ChIP Lysis buffer without protease inhibitors: 50 mM HEPES (pH7.5), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate. Combine 160.6 ml sterile distilled water, 10 ml 1 M HEPES-KOH or HEPES-NaOH (pH 7.5), 7 ml 4 M NaCl, 400 μl 0.5 M EDTA, 20 ml 10% Triton X-100, 2 ml 10% DOC. Store at 4° C.

2. ChIP Lysis buffer + 0.5M NaCl: 50 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate. Combine 142.6 ml sterile distilled water, 10 ml 1 M HEPES-KOH or HEPES-NaOH (pH 7.5), 25 ml 4M NaCl, 400 μl 0.5 M EDTA, 20 ml 10% Triton X-100, 2 ml 10% DOC. Store at 4° C.

3. ChIP LiCl Wash Buffer: 1 mM Tris-HCl, 250 mM LiCl, 0.5% NP-40, 0.5% deocycholate, 1mM EDTA. Combine 167.6 ml sterile distilled water, 2 ml 1M Tris-HCl (pH 8.0), 10 ml 5 M LiCl, 10 ml 10% NP40, 10 ml 10% DOC, 400 μl 0.5 M EDTA, Store at 4° C.

4. TE buffer: 10 mM Tris-HCl (pH 7.4), 1 mM EDTA

5. ChIP TES Buffer: 50 mM Tris-HCl, 10 mM EDTA, 1% SDS. Combine 41.5 ml sterile distilled water, 2.5 ml 1M Tris-HCl (pH 8.0), 1 ml 0.5 M EDTA, 5 ml 10% SDS. Store at room temperature.

6. Roche Complete Protease inhibitor cocktail tablets

7. PMSF: 100 mM in isopropanol. Store at room temperature.

8. 37% formaldehyde

9. 2.5 M glycine

10. Santa Cruz Biotechnology A/G agarose beads

11. 10 mg/ml RNase A

12. 20 mg/ml Proteinase K

13. Ambion 5 μM Glycogen

14. 3 M sodium acetate (pH 5.2)

15. Phenol:chloroform:isoamyl alcohol (25:24:1)

16. Chloroform

17. Phosphate Buffered Saline

2.2. Library preparation

1. Ampure XP PCR purification beads

2. 10 mM Tris-HCl (pH7.5)

3. Double strand adaptor for Illumina sequencing: (NEB or comparable supplier)

4. Dual index primers for library amplification (NEB or comparable supplier)

5. NEB Ultra II End Repair Module

6. NEBNext Ultra II Q5 Hot Start HiFi PCR Master Mix

7. T4 DNA ligase

3. Methods

3.1. Chromatin Immunoprecipitation

Day 1

1. Grow 5 ml overnight culture in liquid medium.

Day 2: Cross-linking, shearing, immunoprecipitation

1. Collect mycelia by vacuum filtration using a Buchner funnel and wash mycelium with 100 mL of PBS.

2. Cross-linking:

   1. Transfer mycelia to 10 ml PBS in a 125-ml Erlenmeyer flask.
   2. Add 270 μl of 37% formaldehyde for a final concentration of 1%.
   3. Incubate on rotating platform for 30 minutes at room temperature.
   4. Add 500 μl 2.5 M glycine to each sample to quench the formaldehyde. Let samples sit at room temperature for 5 minutes.
   5. Collect mycelia by filtration. Wash with PBS.
   6. Transfer mycelia to a 1.5-ml microcentrifuge tube.
3. Lysing cells:

   1. Add 100 μl PMSF and 1 Roche protease inhibitor tablet to 9.9 ml ChIP lysis buffer.
   2. Re-suspend mycelia in 500 μl ice cold ChIP lysis buffer with PMSF and protease inhibitors.
   3. Lyse mycelia by sonicating (see Note 1).
4. Shearing chromatin:

   1. Shear chromatin by sonicating (see Note 1).
   2. Centrifuge samples at 14k RPM for 5 min at 4° C.
   3. Transfer supernatent containing sheared chromatin to a new tube.
   4. Save 20 μl of sheared chromatin extract in new tube and store at −20° C. This will be your input. Use the remaining extract for immunoprecipitation.
5. Equilibration of protein A/G coupled Agarose Beads and overnight binding:

   1. Aliquot 20 μl agarose beads per reaction + 10% total volume into a 1.5-ml microcentrifuge tube.
   2. Spin at 5000 RPM for 1 minute. Discard supernatant.
   3. Resuspend beads in 1 ml ChIP lysis buffer without protease inhibitors.
   4. Repeat steps (b-c).
   5. Resuspend beads in original volume (20 μl/sample + 10%) ChIP lysis buffer without protease inhibitors.
   6. Add 20 μl equilibrated protein A/G beads to each sample. Add 1-3 μl desired antibody.
   7. Incubate overnight at 4° C on rotator to allow antibody binding.

Day 3: Cold washes

1. Spin samples at 5000 RPM for 1 minute to pellet beads. Discard the supernatant by pipetting. Be sure not to disrupt the pellet.

2. Add 1 ml ice-cold ChIP lysis buffer without protease inhibitors to each sample. Incubate for 10 minutes at 4° C on a rotating platform.

3. Spin samples for 1 minute at 5000 RPM at 4° C. Discard the supernatant.

4. Repeat (Steps 2-3).

5. Wash (as in Steps 2-3) with ice-cold ChIP lysis buffer + 0.5M NaCl.

6. Wash (as in Steps 2-3) with ice-cold LiCl wash buffer.

7. Wash (as in Steps 2-3) with ice-cold TE buffer.

8. Collect immunoprecipitated chromatin by adding 62.5 μl TES buffer to each sample. Incubate at 65° C for 10 minutes. Mix by inversion several times during incubation.

9. Spin at 5000 RPM for 1 minute. Transfer supernatant to a new 1.5-ml microcentrifuge tube and save.

10. Repeat (Steps 8-9), saving the supernatant in the same microcentrifuge tube as Step 9.

11. Remove input sample (from Day 2) from −20° C. Add 105 μl TES to each input sample.

12. De-crosslink samples by incubating overnight at 65° C.

Day 4: Final chromatin precipitation

1. Add 125 μl sterile distilled water and 2.5 μl 10 mg/ml RNaseA to samples. Incubate for 2 h at 50° C. Mix samples by vortexing multiple times during incubation.

2. Add 6.25 μl 20 mg/ml Proteinase K. Incubate for 2 h at 50° C. Mix samples by vortexing multiple times during incubation.

3. Add 250 μl phenol:chloroform:isoamyl alcohol to each sample. Mix well by vortexing.

4. Spin at 14k RPM for 5 minutes. Transfer the aqueous layer to a new 1.5-ml microcentrifuge tube.

5. Add 250 μl chloroform. Mix well by vortexing.

6. Spin at 14k RPM for 5 minutes. Transfer the aqueous layer to a new 1.5-ml microcentrigue tube.

7. Add 1 μl glycogen, 25 μl 3M Na-Acetate (pH 5.2), and 865 μl 100% ethanol to each sample. Precipitate overnight at −20° C.

Day 5: Cleanup and elution

1. Retrieve samples from −20° C.

2. Spin at 14k RPM for 10 minutes. Discard the supernatant.

3. Add 300 μl 70% ethanol to each sample.

4. Spin at 14k RPM for 5 minutes. Discard the supernatant.

5. Air dry samples or dry in Speed Vac.

6. Resuspend samples in 25 μl TE.

7. Store at −20° C.

3.2. Library preparation

End Repair

1. Thaw End Repair buffer on ice. Vortex thoroughly to make sure all buffer components are in solution.

2. In a low-bind PCR tube, mix 25.5 μl ChIP DNA, 3 μl 10X End Repair Reaction buffer, 1.5 μl End Prep Enzyme Mix

3. Incubate 30 min @ 20°C, 30 min @ 65°C, Hold @ 4°C

Perform Adaptor Ligation

1. Thaw 10x Adaptor Ligation buffer on ice. Vortex thoroughly to make sure all buffer components are in solution.

2. Dilute double stranded Illumina adaptor to 1.5 μM in 10 mM Tris.

3. Add the following directly to end repair mix: 4 μl of 10x Ligase Buffer with dATP, 2 μl of T4 DNA Ligase, 2 μl of double stranded adaptor, 2 μl of water.

4. Incubate overnight @ 16°C

Bead Cleanup 1

1. Add 40 μl of AmpPure beads and mix by pipetting up and down 10 times.

2. Incubate 5 minutes at room temperature.

3. Place on magnet stand for 5 minutes to clear supernatant.

4. Carefully remove supernatant. Be sure to avoid removing beads.

5. Leaving the tubes on the magnet stand, add 200 μl freshly prepared 80% ethanol.

6. Incubate 30 seconds and remove ethanol wash. Be sure to avoid removing beads.

7. Repeat steps 5 and 6.

8. Air dry beads for 5 minutes on magnet stand with lid open. Be sure not to overdry, as this will make elution difficult.

9. Remove tubes from magnet and elute DNA in 22 μl of 10mM Tris-HCl (pH 7.5-8.0). Mix solution up and down, incubating beads for 5 minutes at room temperature to elute DNA.

10. Place tubes on magnet stand and transfer 20 μl of supernatant to a new PCR tube.

Amplify by PCR

1. In a low-bind PCR tube, combine 20 μl Adaptor ligated DNA fragments, 5ul dual index primer mix containing 10 μM of each primer (use unique dual index combination for each sample you plan to multi-plex), 25ul 2x Q5 Hot start polymerase master mix.

2. Amplify libraries

   1. Denature @ 98°C for 30 sec
   2. For 2 – 12 cycles (Note 3):

      1. 98°C for 10 seconds
      2. 55°C for 30 seconds
      3. 72°C for 60 seconds
   3. 72°C 3 minutes (final extension)
   4. Hold at 10°C

Final Bead Cleanup

1. Add 50 μl of SeraPure beads (1:1 ratio) and mix by pipetting up and down 10 times

2. Incubate 5 minutes at room temp

3. Place on magnet for 5 minutes to clear supernatant

4. Remove supernatant

5. Leaving the tubes on the magnet stand, add 200 μl freshly prepared 80% ethanol.

6. Incubate 30 seconds and remove ethanol.

7. Repeat steps 5 and 6.

8. Air dry beads for 5 minutes on magnet with lid open. Be sure not to overdry.

9. Remove tubes from magnet and elute DNA in 15 μl of 10mM Tris-HCl (pH 7.5-8.0). Mix solution up and down, incubating beads for 5 minutes at room temperature to elute DNA.

10. Transfer 13 μl of supernatant to a new tube. Be sure not to carry over beads, as they will inhibit downstream applications. If carry over occurs, add solution to magnet a second time.

11. Quantify using a bioanalyzer or Qbit fluorometer. If sufficient material is obtained, run 10 – 20 ng of library DNA on a 1.5% agarose gel to confirm correct size distribution and lack of primer dimers.

12. Dilute samples to a concentration of 10 nM. For the 40 Mb Neurospora genome, 50 – 80 individual ChIP-seq samples can be pooled and sequenced on a single flow cell of an Illumina Next-Seq or Hi-Seq instrument. Most ChIP-seq experiments in fungi will require a minimum of 1 – 4 million sequence reads generated using an Illumina sequencing instrument.

3.3. Data analysis

Several analysis options exist for ChIP-seq data. While the specifics of these options may differ based on specific experimental details, the overall approach will require several key steps. Here we will present a general workflow, as well as a small sample of available analysis software.

1. Pre-process sequence reads: Duplicate reads should be removed and Illumina adaptor sequences should be trimmed from any reads that contain them. This is done using FastQC [7] or similar software.

2. Align reads to reference genome using a short read aligner such as bowtie2 [8] or the Burrows-Wheeler Aligner [9].

3. Visualize sequence alignments in a genome browser such as the Broad Integrative Genome Viewer [10] or Gbrowser [11].

4. Perform project specific analyses such as peak calling, analysis of differential enrichment, Motif analysis, etc. HOMER [12], MACS [13], or SICER [14] are commonly used software packages for ChIP-seq analyses.