The overall goal of CARIP-Seq is to survey genome-wide chromatin-associated RNAs. The goal of ChIP-Seq is to evaluate protein-DNA interactions on a global scale in embryonic stem cells. These methods can help answer key questions in the epigenetics and stem cell fields, identify the genome location transcription factors or epigenetic regulator binding or histone modification occupancy or chromatin-associated RNAs in embryonic stem cells.
The main advantage of these techniques is that they allow for the genome-wide unbiased interrogation of RNAs associated with chromatin constituents and global identification of protein DNA interactions. Though these methods can provide insight into embryonic stem cell biology, they can also be applied to other mammalian cell types to study cancer biology or other human diseases. To harvest the embryonic stem cells, wash the cells first with phosphate buffered saline.
Then, detach the cells with 0.25%trypsin, pre-warmed to 37 degrees Celsius. After trypsinizing, resuspend the cells in pre-warmed MEF medium, supplemented with 10%fetal bovine serum. Then, add 37%formaldehyde to the cells in a conical tube to a final concentration of 1%and incubate at 37 degrees Celsius for eight minutes to cross-link the cells.
To stop the fixation reaction, add 1.25 molar glycine pre-chilled at four degrees Celsius to a final concentration of 0.125 molar. Then, place the tube on a rotator and invert it at eight revolutions per minute for five minutes at room temperature. After five minutes, centrifuge the sample at 300g for five minutes at room temperature.
After the centrifugation is over, discard the supernatant. Then, wash the pellet with pre-chilled phosphate buffered saline and again centrifuge the sample. Then, remove the phosphate buffered saline and freeze the cells in liquid nitrogen for a minute.
Once the embryonic stem cells are thawed on ice, resuspend the pellet in 2.5 milliliters of sonication buffer. To study transcription factors or epigenetic factors, resuspend the cell pellet in sonication buffer without sodium dodecyl sulfate. For histone modifications, resuspend the cell pellet in buffer containing sodium dodecyl sulfate.
Next, for chromatin immunoprecipitation, sonicate the chromatin for 14 to 18 cycles at 40%amplitude with 30 seconds on and 30 seconds rest mode. To solubilize the chromatin for the protein factor ChIP, supplement the sonication buffer with 28 microliters of 10%sodium dodecyl sulfate. For protein factors in histone modifications, add 28 microliters of sodium deoxycholate and 0.28 milliliters of 10%Triton X-100 and mix well.
Then, centrifuge the sample at 10, 000g for 10 minutes at four degrees Celsius to pre-clear the chromatin. In a 1.5 milliliter low binding tube, add 40 microliters of A or G magnetic beads. Then, wash the beads with 600 microliters of phosphate buffered saline and transfer the tube with the beads on a rack containing a magnet and incubate for a minute at room temperature.
Then, remove the phosphate buffered saline and again resuspend the beads in 100 microliters of phosphate buffered saline. To the tube, add two to four micrograms of antibody and incubate it by placing on a rotator for 40 minutes at eight revolutions per minute to promote antibody binding to the beads. Then, add 200 microliters of phosphate buffered saline to wash the beads.
After washing, incubate the sonicated chromatin extract on a rotator at four degrees Celsius overnight to immunoprecipitate the chromatin-bound proteins. The next day, wash the beads and incubate with RIPA buffer, RIPA buffer with salt, lithium chloride buffer, Tris-EDTA buffer with Triton X-100, and Tris-EDTA buffer, each for 10 minutes on a rotator. To decrosslink for ChIP, resuspend the beads in 100 microliters of Tris-EDTA buffer.
Then add three microliters of 10%sodium dodecyl sulfate and five microliters of 20 milligrams per milliliter of Proteinase K, and incubate the beads at 65 degrees Celsius overnight. After incubation, the next day, invert the tubes several times, leave them on the magnetic rack, and transfer the supernatant to a fresh tube. Then, wash the magnetic beads by adding 100 microliters of Tris-EDTA buffer, containing 0.5 molar sodium chloride and combine the two supernatants.
To decrosslink for CARIP, resuspend the beads in 100 microliters of Tris-EDTA buffer. Then, add three microliters of 10%sodium dodecyl sulfate and five microliters of 20 milligrams per milliliter of Proteinase K and incubate the beads at 65 degrees Celsius for four to 12 hours. Again, invert the tube several times and place on the magnetic rack to transfer the supernatant to a fresh tube.
Then, wash the magnetic beads by adding 100 microliters of Tris-EDTA buffer, containing 0.5 molar sodium chloride and then combine the two supernatants. Finally, isolate the RNA sample by using phenol chloroform isoamyl alcohol in 25 to 24 to one volume by volume ratio. Next, use DNase to remove DNA from the chromatin associated immunoprecipitated RNA sample.
Then, add 10X DNase buffer to the CARIP sample and adjust the final volume to 1X concentration. Then, incubate the sample at 37 degrees Celsius for 30 minutes. Next, synthesize the first strand of complimentary DNA by setting up a PCR reaction by adding 10.5 microliters of RNA and one microliter of three micrograms per milliliter of random hexamer primers.
To denature the secondary structure, first, incubate the tube at 65 degrees Celsius for five minutes, and then on ice for two minutes. Then, add the master mix to the PCR tube and incubate at 25 degrees Celsius for two minutes. Then, add one microliter of 200 units per microliter of superscript two and run the thermocycler program.
To synthesize the second strand of complimentary DNA, set up the reaction on ice by adding the reagents, buffer, and enzymes. Then, vortex to mix the reagents gently and incubate at 16 degrees Celsius for two hours. After the DNA has been synthesized, purify the complimentary DNA using a PCR purification kit.
Then, defragment the double-stranded DNA, use a water bath sonifier and a 1.5 milliliter tube to share the DNA to a size of 250 to 500 base pairs for three times 10 minutes or four times 10 minutes. Then, prepare the library and perform next generation sequencing. Here, phase contrast images of embryonic stem cells were captured to visualize the stem cell colonies.
In this protocol, the cells were cultured in the presence or absence of mitotically inactivated mouse embryonic fibroblasts with LIF and/or GSK3i in the culture media to prevent the differentiation of the cells. Then, to visualize the efficiency of sonication of the cross-linked embryonic stem cell chromatin, sonicated double-stranded DNA was loaded on a 2%agarose gel. The gel image shows sheared chromatin in the range of 200 to 500 base pairs.
Next, ChIP-Seq libraries were generated and sequenced using a next generation sequencing platform to evaluate genome-wide occupancy of a histone modifying enzyme and histone modifications in embryonic stem cells. From the custom UCSC browser view, enriched H3K4 trimethylation, H3K4 dimethylation, and KDM5B are visible at the core pluripotency regulator gene, POU5F1 and at the HOXC cluster. CARIP-Seq analysis was also performed to evaluate chromatin-associated RNAs in embryonic stem cells.
The custom UCSC browser view shows H4K20 trimethylation associated RNA and H4K20 trimethylation occupancy in embryonic stem cells. From the CARIP-Seq results, it can be interpreted that although the RNA is associated with H4K20 trimethylation, it might not interact with the particular locus. Once mastered, this technique can be done in four days if it is performed properly.
After watching this video, you should have a good understanding of how to survey chromatin-associated RNAs using CARIP-Seq and identify protein DNA interactions on a global scale in embryonic stem cells.
