Enzymatic Chromatin IP Troubleshooting Guide
A. Expected Chromatin Yield
When harvesting cross-linked chromatin from tissue samples, the yield of chromatin can vary significantly between tissue types. The table below provides a range for the expected yield of chromatin from 25 mg of tissue compared to 4 x 106 HeLa cells, and the expected DNA concentration, as determined in Section IV of the protocol. For each tissue type, disaggregation using a Medimachine (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, chromatin processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension. For optimal ChIP results, we recommend using 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per each immunoprecipitation.
|Tissue / Cell||Total Chromatin Yield||Expected DNA Concentration|
|Spleen||20–30 µg per 25 mg tissue||200–300 µg/ml|
|Liver||10–15 µg per 25 mg tissue||100–150 µg/ml|
|Kidney||8–10 µg per 25 mg tissue||80–100 µg/ml|
|Brain||2–5 µg per 25 mg tissue||20–50 µg/ml|
|Heart||2–5 µg per 25 mg tissue||20–50 µg/ml|
|HeLa||10–15 µg per 4 x 106 cells||100–150 µg/ml|
B. Optimization of Chromatin Digestion
Optimal conditions for the digestion of cross-linked chromatin DNA to 150–900 base pairs in length is highly dependent on the ratio of Micrococcal Nuclease to the amount of tissue or number of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific tissue or cell type.
- Prepare cross-linked nuclei from 125 mg of tissue or 2 X 107 cells (equivalent of 5 IP preps), as described in Protocol Sections I, II, and III. Stop after Step 2 of Protocol Section III and proceed as described below.
- Transfer 100 µl of the nuclei preparation into 5 individual 1.5 ml microcentrifuge tubes and place on ice.
- Add 3 µl Micrococcal Nuclease stock to 27 µl of 1X Buffer B + DTT (1:10 dilution of enzyme).
- To each of the 5 tubes in Step 2, add 0 μl, 2.5 μl, 5 μl, 7.5 μl, or 10 µl of the diluted Micrococcal Nuclease, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing.
- Stop each digest by adding 10 µl of 0.5 M EDTA and placing tubes on ice.
- Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
- Resuspend nuclear pellet in 200 µl of 1X ChIP Buffer + PIC. Incubate on ice for 10 min.
- Sonicate lysate with several pulses to break nuclear membrane. Incubate samples 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20 sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator set at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
- Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
- Transfer 50 µl of each of the sonicated lysates to new microfuge tubes.
- To each 50 µl sample, add 100 µl nuclease-free water, 6 µl 5 M NaCl and 2 µl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
- To each RNAse A-digested sample, add 2 µl Proteinase K. Vortex to mix and incubate sample at 65°C for 2 hr.
- Remove 20 µl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker.
- Observe which of the digestion conditions produces DNA in the desired range of 150–900 base pairs (1 to 5 nucleosomes). The volume of diluted Micrococcal Nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to 10 times the volume of Micrococcal Nuclease stock that should be added to one immunoprecipitation preparation (25 mg of disaggregated tissue cells or 4 X 106 tissue culture cells) to produce the desired size of DNA fragments. For example, if 5 µl of diluted Micrococcal Nuclease produces DNA fragments of 150–900 base pairs in this protocol, then 0.5 µl of stock Micrococcal Nuclease should be added to one immunoprecipitation preparation during the digestion of chromatin in Section III.
- If results indicate that DNA is not in the desired size range, then repeat optimization protocol, adjusting the amount of Micrococcal Nuclease in each digest accordingly. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.
C. Troubleshooting Table
|Concentration of the digested chromatin is too low (low chromatin yield).||Not enough tissue or cells were added to the chromatin digestion or cell nuclei were not completely lysed after digestion.||Add additional chromatin to each IP to give at least 5 μg/IP and continue with protocol.|
|Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Some tissues may require processing of more than 25 mg per IP. The amount of tissue can be increased to 50 mg per IP, while still maintaining efficient chromatin fragmentation and extraction|
|Increase the number of sonications following chromatin digestion. Visualize cell nuclei under microscope before and after sonication to confirm complete lysis of nuclei.|
|Chromatin is under-digested and fragments are too large (greater than 900 bp). Large chromatin fragments can lead to increased background and lower resolution.||Too many cells or not enough Micrococcal Nuclease was added to the chromatin digestion.||Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Add less tissue or cells, or more Micrococcal Nuclease to the chromatin digest. See Section B for optimization of chromatin digestion.|
|Tissue or cells may have been over cross-linked. Cross-linking for longer than 10 min may inhibit digestion of chromatin.||Perform a time course at a fixed formaldehyde concentration. Shorten the time of cross-linking to 10 min or less.|
|Chromatin is over-digested and fragments are too small (exclusively 150 bp mono-nucleosome length). Complete digestion of chromatin to mono-nucleosome length DNA may diminish signal during PCR quantification, especially for amplicons greater than 150 bp in length.||Not enough cells or too much Micrococcal Nuclease added to the chromatin digestion.||Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Add more tissue or cells, or less Micrococcal Nuclease to the chromatin digest. See Section B for optimization of chromatin digestion.|
|No product or very little product in the input PCR reactions.||Not enough DNA added to the PCR reaction or conditions are not optimal.||Add more DNA to the PCR reaction or increase the number of amplification cycles.|
|PCR amplified region may span nucleosome-free region.||Optimize the PCR conditions for experimental primer set using purified DNA from cross-linked and digested chromatin. Design a different primer set and decrease length of amplicon to less than 150 bp (see primer design recommendations in Protocol Section VIII).|
|Not enough chromatin added to the IP or chromatin is over-digested.||For optimal ChIP results, add 5 to 10 μg chromatin per IP.|
|No product in the positive control histone H3-IP RPL30 PCR reaction.||Not enough chromatin or antibody added to the IP reaction or IP incubation time is too short.||Be sure to add 5 to 10 μg of chromatin and 10 μl of antibody to each IP reaction and incubate with antibody overnight and an additional 2 hr after adding Protein G beads.|
|Incomplete elution of chromatin from Protein G beads.||Elution of chromatin from Protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.|
|Quantity of product in the negative control Rabbit IgG-IP and positive control histone H3-IP PCR reactions is equivalent (high background signal).||Too much or not enough chromatin added to the IP reaction. Alternatively, too much antibody added to the IP reaction.||For optimal ChIP results, add 5 to 10 µg of chromatin and 10 μl of histone H3 antibody to each IP reaction. Reduce the amount of normal rabbit IgG to 1 μl per IP.|
|Too much DNA added to the PCR reaction or too many cycles of amplification.||Add less DNA to the PCR reaction or decrease the number of PCR cycles. It is very important that the PCR products are analyzed within the linear amplification phase of PCR. Otherwise, the differences in quantities of starting DNA cannot be accurately measured. Alternatively, quantify immunoprecipitations using real-time quantitative PCR.|
|No product in the Experimental Antibody-IP PCR reaction.||Not enough DNA added to the PCR reaction.||Add more DNA to the PCR reaction or increase the number of amplification cycles.|
|Not enough antibody added to the IP reaction.||Typically a range of 1 to 5 μg of antibody are added to the IP reaction; however, the exact amount depends greatly on the individual antibody. Increase the amount of antibody added to the IP.|
|Antibody does not work for ChIP.||Find an alternate antibody source.|
posted March 2008
revised September 2012