All the protocols I have read require upwards of 5 million cells. I am limited to a much smaller number of cells, around 30,000. Can I just scale down a protocol optimized for the high number of cells or is there a protocol specifically for this smaller number of cells? Thanks in advance
I don't know, but would also love to know the answer to this question.
According to my current knowledge, there are no techniques other than those developed from 3C and include: 1) 3C and ChIP version of 3C (ChIP-loop assay), (2) 4C and ChIP version of 4C (enhanced 4C), (3) 5C and 3D assays and (4) Genome conformation capture (GCC) related (Hi-C), ChIP version of GCC as 6C. However, the application to analyze DNA segments by microarray and high-throughput sequencing in the 4C, 5C and Hi-C methodologies has brought the assessment of chromosome interactions to the genome-wide scale.
I have not seen a scaled-down protocol, but it is worth trying. The determining factor, I guess, will be the number of inter-locus interactions governed by your protein of interest within the cell population. You can add sequencing tags to capture all 'captured' interactions, which, arguably, you can scale-up using standard amplification protocols used during ChIP-chip. Here, the chance of your detecting the interaction will be greater. If you are interested in identifying the interaction between two specific loci only, the chance is less. Still, on a conservative scale, if you consider that a third (no reasoning, purely based on speculation) of your cells display the interaction, and a tenth thereof will be captured, and a sixth thereafter will survive the washes and elution, you will end up with 200-odd interactants (considering the genome as diploid). So, before starting the real experiment, I sugest you perform simple PCR for your region of interest taking 10, 20, 200, and 2000 cells--straingt, you do not need to extract DNA prior to PCR (just make sure that the cell suspension is serially diluted in a PCR-compatible buffer just prior to addition to the reaction). You might also want to ChIP your protein from 30000 cells and see if you can detect enrichment at a locus by simple PCR. If you can, there is no reason why the 3C will not work. I shall appreciate readers to point out flaws, if any.
Hi Bradford, write a short e-mail to Job Dekker ([email protected]).
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