Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

1. Standard ChIP Experiments

1. Cell collection and DNA-protein crosslinking.
   1. Grow HeLa-S3 cells to a confluence of 80%-90%. Remove culture medium, wash the dish twice with 10 ml of 1x phosphate buffered saline (PBS), and add Trypsin-EDTA (1x) to the cultured plate. Incubate for a maximum of 2 min to detach the cells from the dish. Collect cells and wash twice with 10 ml PBS.
      NOTE: Longer incubation times will lead to cell damage.
   2. Centrifuge the cells for 5 min at 500 x g and resuspend the cells in 20 ml of PBS. Proceed to count the cells.
   3. Centrifuge the cells for 5 min at 500 x g, discard the supernatant and add 500 μl of PBS. The optimal number of cells for the fixation step is 10 million cells per 500 μl of PBS.
   4. Add 13.5 μl of fresh 37% formaldehyde into each aliquot of 500 μl of cell suspension. Fixate the cells for 8 min at RT.
   5. Add 57 μl of 1.25 M glycine solution to stop the fixation. Incubate for 5 min at RT with constant mixing by gentle vortex. Work on ice from this point onwards.
   6. Centrifuge the cells at 500 x g for 5 min at 4 °C and discard the supernatant without disturbing the cell pellet.
   7. Wash the cells twice with 1 ml PBS. Discard gently the supernatant and keep the cell pellet on ice.
2. Cell lysis and chromatin shearing
   1. Add 10 ml of ice cold lysis buffer iL1 to the cell pellet (1 ml of lysis buffer per 1 million cells is the optimal ratio). Pipette up and down several times and incubate for 10 min at 4 °C with gentle mixing.
   2. Centrifuge the lysate for 5 min at 500 x g and 4 °C. Discard the supernatant.
   3. Add 10 ml of ice cold lysis buffer iL2 to the lysates and mix gently by pipetting up and down. Incubate the lysates for 10 min at 4 °C.
   4. Centrifuge for 5 min at 500 x g and 4 °C and discard the supernatant.
   5. Prepare the complete shearing buffer adding the 200x protease inhibitor cocktail (PIC) to the iS1 shearing buffer. Keep the buffer on ice for 5 min and work on ice afterwards. Add 1 ml of the complete iS1 shearing buffer to each 10 million cells pellet and gently mix by pipetting up and down. Prior to sonication incubate the samples on ice for 10 min to reduce viscosity of the sample.
   6. Shear 300 µl aliquots of chromatin by sonication using a water bath sonicator for 2 to 3 sets of 10 cycles each. A cycle consists of 30 sec “ON” and 30 sec “OFF” on a high power setting. Alternatively, use a pico-sonication device with a shorter sonication time of 5 to 10 cycles of 30 sec “ON”, 30 sec “OFF”. Briefly vortex and spin the tubes between the runs. When using other types of sonicators, follow corresponding manufacturer’s instructions for chromatin shearing.
   7. Centrifuge the sheared chromatin at 16,000 x g for 10 min and collect the supernatant to be used immediately in the IP step. Alternatively, store the chromatin at -80 °C for up to 2 months for future use.
   8. Analyze the chromatin shearing efficiency prior to immunoprecipitation step using 1-1.5% TAE agarose gels or bionalyzer. Optimal chromatin fragment sizes range between 100-600 bp.

2. Low Cell ChIP Experiments

1. Cell collection and DNA-protein crosslinking
   1. Grow HeLa-S3 cells to a confluence of 80%-90%. Remove culture medium, wash the dish twice with 10 ml of 1x phosphate buffered saline (PBS), and add 1x Trypsin-EDTA to the cultured plate. Incubate for a maximum of 2 min to detach the cells from the dish.
      NOTE: Longer incubation times will lead to cell damage.
   2. Collect cells by adding 1 ml culture medium containing serum into a 1 ml centrifugation tube. Count the cells.
   3. Centrifuge the cells for 5 min at 500 x g. Bring the cell number to 10,000 cells per ml of culture medium for the fixation.
   4. Add 27 μl of 36.5% fresh prepared formaldehyde into each tube for the fixation. Invert the tube two or three times and incubate 10 min at RT.
   5. Add 115 μl of 1.25 M glycine solution to the sample, invert the tube two or three times and incubate 5 min at RT. Work on ice from this point onwards.
   6. Centrifuge the cells at 300 x g for 10 min at 4 °C. Discard the supernatant slowly.
   7. Wash cells with 1 ml ice cold HBSS with PIC (200x, final concentration 1x). Invert the tube two or three times to resuspend the cells and centrifuge at 300 x g for 10 min at 4 °C. Discard gently the supernatant and keep the cell pellet on ice.
2. Cell lysis and chromatin shearing
   1. Add 25 μl of complete Lysis Buffer tL1 (Lysis Buffer tL1 + PIC) per 10,000 cells and agitate manually the bottom of the tube to resuspend the cells. Incubate on ice for 5 min.
   2. Add 75 μl of complete HBSS (HBSS + PIC) buffer into each aliquot containing 10,000 cells.
   3. Shear 100 μl aliquots of 10,000 cells by sonication for 5 sets of 5 cycle each. A cycle consists of 30 sec “ON” and 30 sec “OFF” on the high power setting. Alternatively, use a pico-sonication device used with a shorter sonication time of 5 cycles of 30 sec “ON”, 30 sec “OFF”. Optimal chromatin fragment sizes range between 100-600 bp. Note that chromatin preparations, cell types and different sonicators require separate shearing optimization experiments.
   4. Centrifuge the sheared chromatin at 14,000 x g for 10 min to discard the insoluble material and collect the supernatant to be used immediately in the IP step. Alternatively, store the chromatin at -80 °C for up to 2 months for future use.
   5. Analyze the chromatin shearing efficiency prior to immunoprecipitation step using 1-1.5% TAE agarose gels or the bionalyzer. Treat samples with RNase prior to agarose gel analysis in order to improve the visual assessment of shearing. Optimal chromatin fragment sizes range between 100-600 bp.

3. Chromatin Immunoprecipitation and Library Prep

1. For standard automated ChIP experiments
   1. Add 120 μl of ChIP Buffer H (ChIP Buffer H + PIC) to 100 μl sheared chromatin. Use 200 μl for the IP and keep 2 μl to 20 μl as input sample.
   2. Select the automated ChIP 200 μl protocol in the automation instrument. The throughput of the protocol is 1 to 16 samples per run.
   3. Run an automated ChIP experiment using, chromatin corresponding to 1-2 million cells, 1-2 μg of anti-H3K79me3, -H3K27me3, -H3K4me3, -H3K4me2, -H3K9ac, -H3K9/14ac, -H3K36me3 and H3K9me3 ChIP-seq grade rabbit polyclonal antibodies. Optimal antibody amounts vary depending on the histone modification and the affinity and specificity of the corresponding antibody.
      1. Use equal amounts of non-immune rabbit IgG as an isotype control antibody. Alternatively, use uncoated beads or specific blocked antibody as ChIP controls. Add 20 μl of Protein-A coated magnetic beads for each reaction.
   4. Use the automated histone ChIP-seq kit reagents to perform automated ChIP experiments with anti-H3K79me3 and -H3K4me2 polyclonal antibodies. Use the ideal ChIP-seq kit reagents to perform ChIP experiments with anti-H3K27me3, -H3K4me3, -H3K9ac, -H3K9/14ac, -H3K36me3 and -H3K9me3.
   5. Select the automated ChIP protocol following the software instructions implemented in the automation device. Set the ChIP experimental parameters to 4 hr for the antibody coating step and 15 hr for the immunoprecipitation step. The reverse crosslinking step takes place in the automated instrument at 65 °C for 4 hr.
   6. Purify the reverse cross-linked DNA on the automated system. Select automated protocols for DNA purification with a protocol or kit using magnetic bead based DNA purification. Elute the DNA in 25 μl of water.
   7. Quantify the immunoprecipitated DNA by extracting 10% of the immunoprecipitated DNA. The immunoprecipitated DNA yield depends on the quality of the chromatin and antibody, cell type and the target histone modification. Quantify the DNA using an assay kit according to manufacturer’s instructions.
   8. Analyze the quality of the immunoprecipitated DNA by quantitative PCR using primers for at least 1 positive and 1 negative control genomic regions. Do not use more than 10% of the total immunoprecipitated DNA for assessing ChIP enrichments.
      1. Prepare the qPCR reactions. Add 10 μl of a 2x SyberGreen qPCR master mix, 1 μl of primer mix, 1-5 μl of immunoprecipitated or input DNA and sterile water up to 20 µl final reaction volume. The qPCR program includes an initial denaturation step at 95 °C for 5-10 min depending on the provider of the Taq polymerase and the annealing temperatures should be set according to the primers selected.
   9. Use automated library prep protocols compatible with the commercially available Illumina ChIP-seq library preparation reagents to build the libraries using both ChIP DNA as well as the saved input DNA from the same chromatin preparation. Use 10-20 ng of immunoprecipitated DNA from each antibody for library preparation. Prepare up to 16 automated libraries per run.
   10. Sequence the libraries and generate clusters according to Illumina manufacturer’s instructions. Perform primary bioinformatics analysis (cluster filtering, base calling, etc.) following the standard Illumina pipeline, filter and align the reads to the latest human genome assembly (current version is GRCh38) with the ELAND aligner. Use SICER⁷ or MACS⁸ for peak calling and perform the downstream analyses of peaks with Homer⁹, BEDTools¹⁰ or preferred software.
2. For low cell number automated ChIP experiments
   1. Add 120 μl of complete ChIP buffer tC1 (ChIP Buffer tC1 + PIC) to 100 μl sheared chromatin. Use 200 μl for the IP and keep 20 μl as input.
   2. Select automated ChIP 200 μl protocol in the automation system. The throughput of the protocol is 1 to 16 samples per run.
   3. Run an automated ChIP-seq experiment using automated ChIP reagents and ChIP grade antibodies optimized to work on low chromatin quantities. Use chromatin corresponding to 10,000 cells and 100,000 cells, 0.5 μg of anti-H3K27me3, 0.25 μg -H3K4me3, 0.1 μg -H3K27ac, 0.25 μg -H3K9me3 rabbit Premium ChIP-seq grade rabbit polyclonal antibodies. Optimal antibody amounts vary depending on the histone modification and the affinity and specificity of the corresponding antibody.
      1. Use equal amount of non-immune rabbit IgG as an isotype control antibody. Alternatively, use uncoated beads or specific blocked antibody as ChIP controls. Add 10 μl of Protein-A coated magnetic beads for each reaction.
   4. Select the automated ChIP protocol following the software instructions implemented in the automation device. Set the ChIP experimental parameters to 4 hr for the antibody coating step and 15 hr for the immunoprecipitation step. The reverse crosslinking step takes place in the automated instrument at 65 °C for 4 hr.
   5. Purify reverse cross-linked DNA using spin columns following manufacturer’s instructions and elute in volumes from 6 μl to 25 μl of water.
   6. Quantify the DNA using a commercial assay kit. Analyze the results by qPCR using primers for positive and negative control regions to evaluate the ChIP quality.
   7. Use a library preparation kit with optimized library preparation reagents to prepare libraries with low DNA quantities. Use 30 pg and 300 pg of ChIP DNA (corresponding to 10,000 and 100,000 cells experiments respectively) for library preparation. Prepare libraries using the automated protocol compatible with library preparation reagents. The automated library prep throughput is 1 to 48 libraries per run.
   8. Following the end repair of the double-stranded DNA templates, ligate the cleavable stem-loop adaptors containing the sequencing primer sites. Following the DNA extension step, amplify the sample with the high fidelity amplification method described in the library preparation kit protocol.
   9. After library amplification, quantify and purify the libraries following the library preparation kit guidelines. Note that size selection after purification is not necessary.
   10. Sequence the libraries and generate clusters according to the manufacturer’s instructions. Perform primary bioinformatics analysis (cluster filtering, base calling, etc.) following the standard manufacturer pipeline, filter, and align the reads to the latest human genome assembly (current version is GRCh38) with the ELAND⁷ aligner. Use SICER⁸ or MACS⁹ for peak calling and perform the downstream analyses of peaks with Homer, BEDTools¹⁰, or any preferred software.

Figure 1. Screenshots of the software showing how to set up automated ChIP experiments in the IP-Star Compact. The software provides the flexibility to select the amount of samples per run as well as to change key experimental parameters (Antibody Coating, IP and washes) according to the researcher needs. The automated procedure allows testing different conditions in parallel (i.e., different types and amounts of antibodies, different types and amounts of cells and even different types and amounts of magnetic beads in the same run. Please click here to view a larger version of this figure.

Figure 2. Screenshots of the software showing how to set up automated library preparation for next generation sequencing using the library kit in the automation system. Please click here to view a larger version of this figure.