Chromatin Immunoprecipitation and sequencing can be used to characterise protein-DNA interactions. It works by cross-linking proteins to DNA, shearing the DNA and then pulling down the protein of interest. Any DNA that is bound to the protein of interest can then be identified by sequencing in order to characterise the DNA binding sites for the protein of interest.
In order to carry out a ChIP-Seq experiment, a specific antibody for the protein of interest is required. Alternatively, a strain expressing the protein of interest with a tag fused to one terminus (e.g. a 3x FLAG-TAG) to allow for immunoprecipitation of the protein-DNA complexes.
- Lysis Buffer – 10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mg/ml lysozyme
- IP Buffer – 100 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% v/v Triton-X
- TE Buffer – 10 mM Tris-HCl pH 8.0, 1 mM EDTA
- Elution Buffer – 50mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS
- EB Buffer – 10 mM Tris-Cl pH 8.5
- Sampling: Cultures can be grown in either liquid culture or on solid agar plates (on cellophane discs). For sampling, harvest liquid cultures by centrifugation or removing cellophane discs and biomass from the plate at the desired time point.
Optional: At this point, a small sample can also be taken for western blot to confirm expression of the desired protein using an antibody that is specific to the protein of interest of the tag.
- Cross-link proteins to DNA: Wash pellet or submerge disc in 10 ml of a 1% (v/v) formaldehyde solution for 20 minutes at room temperature to cross-link proteins to DNA. Discs/pellets are then washed in 10 ml of 0.5 M glycine for 5 minutes before the biomass is harvested. Wash cells twice in 25 ml ice-cold PBS (pH 7.4). Pellets can either be flash frozen in liquid nitrogen for storage at -80°C or used straight away.
NB: From this point onwards, protease inhibitors should be added to all buffers and work should be conducted on ice where possible.
- Cell Lysis: Resuspend pellets in 1-2 ml lysis buffer and incubate at 37°C for 30 minutes.
- Fragmentation of DNA: Add 1 ml IP buffer and mix by pipetting. Sonicate twenty times at 50 Hz, 10 seconds/cycle with at least 2 minutes incubation on ice after each sonication cycle.
Mix 25 μl of crude lysate with 75 μl TE buffer and extract with 200 μl phenol:chloroform. Separate this extracted DNA into two; 25 μl should be transferred to a fresh tube, 2μl RNaseA (1mg/ml) added, incubated at 37°C for 30 minutes. This sample should them be analysed by gel electrophoresis to determine whether the DNA has fragmented to the required sizes for sequencing (usually between 200 and 100bp fragments).
Once fragmentation is confirmed, the remaining crude lysate can be cleared by centrifugation at 4000 rpm for 15 minutes at 4°C.
- Immunoprecipitation: For binding, prepare 500 μl Anti-FLAG M2 beads by washing in 2.5 ml 0.5 IP buffer and add 40 μl to the cleared lysate. Incubate on a vertical rotor overnight at 4°C.
Harvest beads gently by centrifugation and wash 4 times in 0.5 IP by incubating on a vertical rotor for 10 minutes at 4°C. Remove as much buffer as possible without removing the beads.
N.B: For this step, it can be useful to prepare “thin” pipette tips by squeezing the end of tips with forceps – this will ensure that fewer beads are lost during the wash process.
- Elution: Add 100 μl elution buffer and incubate at 65°C overnight.
Remove ~100 μl elution buffer and SAVE IT (this is your sample!). Add another 50 μl elution buffer to the beads and incubate at 65°C for a further 5 minutes and remove so that the total final sample is ~150 μl.
- DNA Extraction: Add 2 μl 10 mg/ml proteinase K and incubate at 55°C for 1.5 hours. Add 150 μl phenol-chloroform vortex and centrifuge at full speed for 10 minutes. Remove the aqueous layer and purify on a Qiaquick column (Qiagen) according to the manufacturers protocol. Elute in 50 μl EB buffer – take ~10 μl for quantification (by nanodrop, Qubit etc.) and snap freeze the remaining ~40 μl sample in liquid nitrogen and store at -20 °C for sequencing.
This protocol has successfully been used in S. coelicolor, S.venezualea and S. formicae (see publications below).
https://mbio.asm.org/content/mbio/7/2/e00523-16.full.pdf https://www.frontiersin.org/articles/10.3389/fmicb.2017.01145/full https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000524
Protocol adapted for Actinobase by Rebecca Devine, The University of East Anglia