Chromatin immunoprecipitation sequencing (chip-seq)
Chromatin immunoprecipitation sequencing (chip-seq)
Summary
Chromatin immunoprecipitation assay (ChIP) is currently the only method to study DNA-protein interactions in vivo, which mainly includes ChIP-qPCR and ChIP-seq. ChIP-seq can efficiently detect DNA regions that interact with histone modifications, transcription factors, etc. on a genome-wide scale.
Principle
The genomic DNA of eukaryotes exists in the form of chromatin. The target protein and DNA are cross-linked with formaldehyde, and the chromatin is fragmented by ultrasonication or nuclease digestion; then antibodies are used to specifically precipitate and separate histones or transcription factors and their bound chromatin, i.e., chromatin immunoprecipitation products; the target protein is separated from the DNA by proteolytic cross-linking, and the enriched DNA fragments are purified and detected and quantified by next-generation sequencing (NGS) technology. DNA fragments enriched by Next Generation Sequencing (NGS).
Operation method
Chromatin immunoprecipitation sequencing (chip-seq)
Principle
The genomic DNA of eukaryotes exists in the form of chromatin. The target protein and DNA are cross-linked with formaldehyde, and the chromatin is fragmented by ultrasonication or nuclease digestion; then antibodies are used to specifically precipitate and separate histones or transcription factors and their bound chromatin, i.e., chromatin immunoprecipitation products; the target protein is separated from the DNA by proteolytic cross-linking, and the enriched DNA fragments are purified and detected and quantified by next-generation sequencing (NGS) technology. DNA fragments enriched by Next Generation Sequencing (NGS).
Materials and Instruments
Formaldehyde, glycine, pre-cooled PBS, ChIP lysis buffer, elution buffer, NaCl, RNase, Proteinase K, Tris-EDTA (TE), 100 bp DNA marker, 1.5% agarose gel, RIPA buffer, primary antibody, Protein A/G beads, low-salt wash buffer, high-salt wash buffer, lithium chloride wash buffer. buffer, high salt wash buffer, lithium chloride wash buffer. Move 1. DNA-protein cross-linking and cell collection Crosslink proteins and DNA with formaldehyde; the crosslinking procedure is time dependent and needs to be optimized. Tip: The recommended crosslinking time for samples is 2-30 minutes. Excessive crosslinking reduces antigen accessibility and sonication, and epitopes may be masked. Add glycine to seal the formaldehyde and terminate the cross-linking reaction. 1.1 First, prepare 2×107~1×108 cells. Add formaldehyde directly to the medium drop by drop to a final concentration of 0.75%, and then gently rotate for 10 minutes at room temperature to induce cross-linking of proteins and DNA. 1.2 Add glycine to the medium to a final concentration of 125 mM and incubate for 5 minutes at room temperature with shaking. 1.3 Rinse cells twice with 10 mL of ice-cold PBS. 1.4 Add 5 mL of ice-cold PBS, scrape cells, and transfer to a centrifuge tube. 1.5 Centrifuge at 1,000 g for 5 minutes at 4 °C. 1.6 Carefully aspirate the supernatant and resuspend the precipitate in ChIP Lysis Buffer (750 μL per 107 cells) and incubate on ice for 10 minutes. 2. 2. Sonication of cell lysates to separate chromatin 2.1 Sonicate the lysate to shear the DNA and ensure that the average fragment size of the DNA is 200 to 1,000 bp. The sonication time should be optimized. 2.2 After sonication, centrifuge the lysate at 8,000 g for 10 minutes at 4 °C and remove the supernatant into a new tube. 3. Calculate the DNA concentration and determine the amount of DNA in the lysate. 3. Calculate the DNA concentration and determine the size of DNA fragments. The sonicated chromatin sample can be used to calculate the DNA concentration of the subsequent IP and to electrophoretically determine the size of the DNA fragments. 3.1 Take 50 µL of the sonicated sample and determine DNA concentration and fragment size. 3.2 Add 70 µL of Elution Buffer to 50 µL of chromatin. 3.3 Add 4.8 µL of 5 M NaCl and 2 µL of RNase A (10 mg/mL) and incubate overnight at 65 °C with shaking. 3.4 Add 2 µL of Proteinase K (20 mg/mL) and incubate at 60 °C for 1 hour with shaking. 3.5 Purify DNA using PCR purification kit or phenol:chloroform extraction. 3.6 To determine the DNA concentration, take 5 μL of purified DNA, transfer it to a tube containing 995 μL of TE, dilute it 200-fold, and read the OD260. the DNA concentration (μg/mL) is OD260 × 10 000. this is used to calculate the DNA concentration of the prepared chromatin. The DNA is purified by electrophoresis in a 1.5% agarose gel with a 100 bp DNA marker to determine the fragment size. 4. 4. Chromatin immunoprecipitation (IP) 4.1 Use the chromatin prepared in step 2.2. It is recommended that approximately 25 μg of DNA be used per IP. dilute each sample with RIPA buffer at a ratio of 1:10. Set up an IgG antibody control and a control with magnetic beads only. Take 50 µL of chromatin as an input sample and store at -20 °C. 4.2 Add primary antibody to all samples (except the bead-only control) and incubate for 1 hour at 4 degrees Celsius with rotation. The amount of antibody should be determined experimentally in advance; in general, add 1~10 μg of antibody per 25 μg of DNA (the best antibody is the one validated by ChIP, followed by IP validation; refer to the manufacturer's recommendation for the amount of antibody). 4.3 Prepare protein A/G beads (choose the appropriate beads according to the experiment) 4.4 Add 60 µL of Protein A/G beads to all samples and spin at 4 °C overnight. 4.5 Centrifuge at 2,000 g for 1 minute to remove supernatant. 4.6 Wash as follows: one wash in Low Salt Wash Buffer, one wash in High Salt Wash Buffer, and one wash in Lithium Chloride Wash Buffer. After each wash, centrifuge at 2,000 g for 1 minute to remove the supernatant. 5. Elution and cross-linking 5.1 Add 120 μL of Elution Buffer to the Protein A/G beads to elute the DNA, and slowly vortex for 15 minutes at 30 °C. 5.2 Centrifuge at 2000 g for 1 minute. 5.2 Centrifuge at 2000 g for 1 minute and transfer the supernatant to a new tube. 5.3 Add 4.8 µL of 5 M NaCl and 2 µL of RNase A (10 mg/mL) and incubate overnight at 65 °C with shaking. 5.4 Add 2 µL of Proteinase K (20 mg/mL) and incubate at 60 °C for 1 hour with shaking. 5.5 DNA can be purified using PCR purification kit or phenol-chloroform extraction. 6. 6. ChIP-seq analysis of DNA levels When using the ChIP-seq method, DNA levels are analyzed by sequencing. The DNA produced using this protocol is suitable for use as input for the preparation of sequencing libraries. Caveat 1. Protein A and G beads have different affinities for different immunoglobulin isoforms of various genera and are selected based on the IP antibody used. 2. If a high background is observed, additional washing steps may be required. Alternatively, the Protein A/G Beads can be pre-incubated for 1 hour prior to step 4.2 to remove non-specific binding to the beads. Transfer the supernatant (sonicated chromatin) to a new tube and incubate the antibody and beads as in step 4. 2. Common Problems 1. Why use RNase A? When using PCR purification kits, high levels of RNA can interfere with DNA purification, so it is necessary to treat the samples with RNase A. Otherwise, the product will be greatly reduced when the column is saturated. Otherwise, the product will be greatly reduced when the purification column is saturated with adsorbent. 2. Why use Proteinase K? Treatment of the sample with Protease K breaks the peptide bond between the neighboring fat and the carboxyl group of the aromatic amino acid. This breaks the cross-links between protein and DNA and facilitates DNA purification. 3. Chromatin-digested fragments that are too large (>1,000 bp) or too small (<150 bp). Excessive cross-linking. Crosslinking for more than 10 minutes may inhibit chromatin digestion. Not enough cells added to chromatin digestion. Different cell lines require different sonication times. 4. 4. Equal amounts of product in negative control IgG-IP and experimental group antibody IP PCR reactions. Too much or not enough chromatin is added to the IP reaction. Alternatively, too much antibody was added to the IP reaction. 5. 5. No product in the IP PCR reaction with the experimental antibody. Not enough DNA was added to the PCR reaction. Not enough antibody added to the IP reaction. Antibody is not suitable for IP. For more product details, please visit Aladdin Scientific website.