The overall goal of this protocol is to unbiasedly identify combinatorial chromatin state patterns in tumor tissues and cancer cell lines. This method could help answer key questions in the cancer epigenetics field such as how a barren histone modification states could contribute to tumor genesis. The main advantage of this technique is that multiple histone modifications, as well as multiple samples could be processed both simultaneously, as well as efficiently.
First, dissociate 50 milligrams of melanoma tissue manually and two milliliters of Hank's Balanced Salt Solution or HBSS, using a sterile razor blade. Mince the tissue into three or four millimeter pieces for approximately five minutes in a sterile tissue culture dish. Transfer the tissue solution to a dissociator tube and add another eighth milliliters of HBSS.
Further dissociate the tissue, using a dissociator, until homogenized. Next, crosslink the tissue, adding 200 microliters of 16%formaldehyde per three milliliters of HBSS. Using a mixer, shake the mixture at 10 rpms for exactly 10 minutes at 37 degrees Celsius.
After removing the sample from the incubator, add 200 microliters of tumoral glycine per three milliliters of sample and shake the mixture at 10 rpms for five minutes at 37 degrees Celsius. Spin the sample at 934 times g for five minutes at four degrees Celsius using a bench top centrifuge. When finished, remove the supernatant and add five milliliters of ice cold PBS.
Then, centrifuge the sample again at 934 times g then remove the supernatant. Add 300 microliters of ChIP harvest buffer with protease inhibitors per 50 milligrams of tissue and lyse the samples for 30 minutes on ice. While the cells are lysing, turn on the water bath disruptor and associated cooling system and allow the temperature to reach four degrees Celsius.
Place the sonicator tube in the water bath disruptor and sonicate the melanoma tissue for 60 cycles at 30 seconds on and 30 seconds off to obtain chromatin fragments of 200 to 600 base pairs. For eight milligrams of melanoma tissue, incubate three micrograms of each histone antibody with 20 microliters of protein g magnetic beads and 100 microliters of binding blocking buffer for two hours at four degrees Celsius with rotation, using a tube revolver. After sonication, dilute the sample five times using ChIP dilution buffer to bring the SDS concentration down to 0.1%After incubation of the antibody and protein g magnetic beads is complete, remove the ChIP dilution buffer and re suspend the beads in 240 microliters of the fresh buffer.
Then, place 20 microliters aliquots of the beads into 12 separate tubes, each of which will be used for a separate tissue sample. Aliquot the sonicated material evenly to the protein g beads with specific antibody and rotate the tubes using a tube revolver overnight at four degrees Celsius. On the morning after reverse cross linking, transfer the antibody protein solution to a 96 well plate and place it on a magnetic stand.
After allowing the beads to adhere for at least 30 seconds, remove the supernatant. Wash the beads five times with 150 microliters of ice cold RIPA wash buffer using a multi channel pipette. Move the magnet continuously for 30 seconds per wash, then remove the supernatant.
After the washing steps, add an elution buffer master mix, containing 44 microliters of direct elution buffer, one microliter of Rnase and five microliters of protenase k per chip and input the sample for reverse cross linking. Incubate the samples using a PCR thermal cycler for four hours at 37 degrees Celsius, four hours at 50 degrees Celsius and eight to 16 hours at 65 degrees Celsius. On the next morning, place the samples back on the magnet and transfer the supernatant to a new 96 well PCR plate which contains the amino precipitated DNA.
Add 2.3 x paramagnetic beads to the solution and carefully pipette up and down 25 times using a multi channel pipette. Following this, incubate at room temperature for four minutes. Then place the samples back on the magnet and incubate at room temperature for another four minutes.
After discarding the supernatant, add 150 microliters of 70%ethanol to the samples and incubate at room temperature for 30 seconds without disturbing the beads. After allowing the beads to dry, remove the samples from the magnet and add 30 microliters of 10 millimolar traceal. Mix by pipetting 25 times.
After incubating the samples at room temperature, place them back on the magnet and incubate at room temperature for another four minutes. Transfer 20 microliters of each sample to a new 96 well plate for library preparation. After PCR amplification, remove the samples and bring the total volume to 100 microliters using nucleus free water.
Perform double sided paramagnetic size selection by first adding 55 microliters of beads to each sample and mixing 25 times with a pipette. Then incubate at room temperature for four minutes. Place the samples back on the magnet and incubate at room temperature for another four minutes to attain large fragments on the beads that are unsuitable for sequencing.
After incubation, transfer the supernatant to a new 96 well PCR plate. Add another 25 microliters of paramagnetic beads and mix by pipetting 25 times and incubate at room temperature off the magnet for four minutes. After incubating at room temperature, place the samples back on the magnet and incubate at room temperature for four minutes.
Then discard the supernatant. While leaving the samples on the magnet, add 150 microliters of 70%ethanol and incubate at room temperature for 30 seconds without disturbing the beads. Remove the ethanol and repeat the wash.
After allowing the beads to dry, remove the samples from the magnet and add 25 microliters of 10 millimolar traceal pH 8.0. Mix by pipetting 25 times and incubate at room temperature, off the magnet for four minutes. After incubating at room temperature, place the samples back on the magnet and incubate at room temperature for four minutes.
Now, quantify the libraries using a high sensitivity DNA electropherogram instrument before multiplexing to ensure the sizes are suitable for sequencing. This protocol allows immunoprecipitation of frozen tumor tissues and cell lines that can be performed on dozens of samples and parallel using a high throughput method. Chromatin fragments should range between 200 and 1000 base pairs for optimal immunoprecipitation.
Purified ChIP DNA should be quantified before proceeding to library preparation. Completed libraries suitable for multiplexing and next generation sequencing should range between 200 and 600 base pairs and be devoid of any primer dimers. Upon post sequencing, there are many quality control metrics that should be met before proceeding to further steps of the pipeline, such as macs peak calling and chromHmm analysis.
ChIP samples should be enriched over input DNA and each histone modification should display a distinct profile representing a specific component of the epigenetic landscape. The chrome HMM algorithm uses a Multivariate Hidden Markov Model to identify the most prominent reoccurring combinatorial and spacial chromatin patterns based on the histone modifications studied. Using these six modifications, chrome HMM identifies functionally distinct chromatin states representing both repressive and active domains, such as polycomb repression, heterochromatic repression, active transcription, and active enhancers.
Once mastered, this technique can be done in three to four days, if it's performed properly. While attempting this procedure, it's important to determine optimal sonication conditions and specification for antibodies before continuing to library preparation and data analysis. For learning this procedure, other methods, such as RRS sequencing and chromosome confirmation capture can be performed to determine coalition of chromatin state parts with gene expression and higher chromatin structure.
After the experiment, the technique has paved way for researchers in cancer epigenetics field to explore chromatin state patterns in patient tumor samples. After watching this video, you should have a good understanding of how to process and disassociate tumor tissues, perform chromatin immunoprecipitation, prepare libraries for nextgen sequecing, process post sequencing data and gain insights into initial steps of downstream analysis. Don't forget that when working with formaldehyde, it can be extremely hazardous and precautions, such as an appropriate fume hood, should always be taken when performing this procedure.
