This experimental method permits the long-term storage of the reusable single cells for epigenomic testing. It also allows the analysis of many epigenetic marks and transcription factors in the same single cell. In current methods for single-cell epigenome analysis, the same single cells cannot be reanalyzed.
However, with reusable single cells, the same single cells can be reanalyzed many times. This technique is useful for diagnosis and design of epigenetic therapy, particularly when patient cells are limited in number. This method is most suitable to the study of the epigenome, the regulation of gene expression, and other systems as long as specific antibodies for epigenetic marks are available.
When you first try this technique, we recommend that you practice on samples that are not too precious, and you validate the experiment by using shallow sequencing such as MiSeq or iSeq 100. To begin, in a clean laminar flow hood, mix one milliliter of cell suspension and 199 milliliters of one millimolar EDTA PBS containing an intercalator dye for DNA. After mixing, transfer 200 microliters of the cell suspension to each well of the flat bottom 96-well plate.
Put the cover onto the 96-well plate and scan the plate using a scanning microscope. Next, tilt the plate and wait for a few minutes until the single cell has sunk to the lower edge of the well. Then place the pipette tip on the bottom corner and transfer the single cell and the buffer to a PCR tube.
Check the well using a fluorescence microscope to confirm the transfer. If a single cell is still in the well, add 200 microliters per well of one millimolar EDTA PBS containing an intercalator dye for DNA and repeat the transfer. After the transfer, place a cover on the 96-well PCR plate and centrifuge the plate using a swing rotor without a break.
After centrifugation, place the plate on the deck of an automatic liquid handling robot. Next, drag and drop the supplemental code one to the software window of the liquid-handling robot and run the program to remove 195 microliters of the supernatant with a very slow pipetting speed. Add 50 microliters of 4%TEMED or mineral oil and incubate the plate overnight at room temperature.
The next day, remove the mineral oil by pipetting and wash the cells five times with 200 microliters of TP magnesium negative buffer. After the last wash, remove the supernatant, add 15 microliters of annealing buffer, and incubate on ice for one hour. After incubation, place the PCR tubes on a thermal cycler and heat at 94 degrees Celsius for three minutes.
Then transfer the tubes to an ice-cooled metal block and incubate for two minutes. Next, add four microliters of magnesium sulfate-sodium chloride, DNTP mix and mix by vortexing at a maximum speed. Then add one microliter of BST polymerase large fragment, mix by vortexing, and incubate for four hours on a shaker.
After incubation, place the PCR tube on a thermal cycler and run a four-hour or overnight program as described in the text. For antibody binding, add 1.625 microliters of sodium chloride EDTA BSA solution to the tube. Mix by vortexing at low speed and incubate the tube on ice for one hour.
Then add 0.1 micrograms per milliliter of antibody and control IgG conjugated with the antibody probe. After adding the antibodies, incubate the tubes on ice with gentle shaking. The next day, wash the cells twice with 200 microliters of TPM-T buffer on ice.
Then remove the supernatant and wash once with T4 DNA ligase buffer. Next, add 19 microliters of ligation adapter solution and incubate for one hour at 25 degrees Celsius. Then, add one microliter T4 DNA ligase and mix the tube by vortexing at medium speed.
Place the tube on a thermal cycler and run the proximity ligation program. After the run, wash the cells twice with 200 microliters of BST magnesium negative EDTA positive buffer and store the cells overnight at four degrees Celsius. The following day, wash the cells twice with 200 microliters of BST magnesium negative EDTA negative buffer, and add 20 microliters of a mixture containing BST, DNTPs, and magnesium sulfate.
Then mix with a vortex mixer and incubate the tube for four hours on an orbital shaker. After incubation, place the tube on a thermal cycler and run the full extension program as described in the text manuscript. Next, for multiple displacement amplification, add 0.4 microliters of 100 micromolar second random primer, and mix by vortexing.
Then, incubate the tube for two hours on an orbital shaker before placing the tube on a thermal cycler for heating at 94 degrees Celsius. After three minutes, place the tubes in an ice-cooled metal block and add one microliter of BST DNA polymerase. Vortex the tubes at slow speed.
Then place the tubes on a thermal cycler to run the multiple displacement amplification program. After the program, transfer approximately 20 microliters of supernatant to a PCR tube, and store it at minus 80 degrees Celsius. Next, add 20 microliters of 0.05%TWEEN 20 and 0.1X TE buffer to a PCR tube containing the reusable single cell, and incubate the tube overnight at four degrees Celsius.
The following day, collect the supernatant and combine it with the supernatant collected previously. Then, soak the reusable single cell and TBS buffer containing 50%glycerol, five millimolar EDTA, 0.5%BSA, and 0.05%TWEEN 20, then incubate it for 30 minutes on an orbital shaker. After incubation, store the reusable single cell at minus 20 degrees Celsius until further use.
Finally, add 40 microliters of Exo negative master mix, and place the tube on a thermal cycler to run the program as described in the text manuscript. K562 reusable single cells were generated using this protocol. Single cells were embedded in the outer layer of the polyacrylamide bead and visualized using an intercalator dye for DNA staining.
DNA products before and after BciVI digestion are shown here. The restriction enzyme digestion generated the expected 19 to 20, 31 to 32, 49, and greater than 49 base pair fragments. Further, the constructed DNA library was analyzed by capillary electrophoresis for the desired products.
The sequencing results indicated that the unique mapped reads from anti-histone H3 acetylated lysine 27 and anti-histone H3 trimethylated lysine 27 were significantly more numerous than from the control IgG. The REpi sequencing signals were evaluated by comparing REpi sequencing data with bulk ChIP sequencing data. On average, approximately 91%of the histone H3 acetylated lysine 27 signals from REpi sequencing overlapped with histone H-3 acetylated lysine 27 peaks in bulk ChIP sequencing.
The precision of REpi sequencing for the inactive histone mark, histone H3 trimethylated lysine 27 was 52%The sensitivity of REpi sequencing was analyzed to measure how many peaks of bulk ChIP sequencing were detected by REpi sequencing reproduced reads. The sensitivity of REpi sequencing was 55.30%in histone H3 acetylated lysine 27 and 50.94%in histone H3 trimethylated lysine 27. Please make sure you use DNS-free reagents.
Also, all operations that involve the opening tube leads or reagent leads should be performed in a clean hood. DNA products of this procedure are sequenced and then mapped to the genome to reveal which histone modifications are present, in which regions of the genome in single cells. This technology made it possible to analyze multiple epigenetic marks in the same single cell and paved the way for multiomics analysis in any single cell.
