Chilly, wintry temps have us longing for palm trees, sandy beaches, and the limbo call of “How low can you go?” Researchers in Norway and Germany have the answer—and a new ChIP-seq method that allows them to analyze genome-wide DNA-protein interaction data for samples that have very few cells, without culturing (which could alter epigenetic mods) and excessive PCR runs.
Current ChIP-seq methods require 1-20 million cells per IP, which can be impossible to obtain when using real-world patient samples or stem cells. There’s been quite a bit of improvements to low cell number ChIP-seq protocols, but they’re usually based on formaldehyde crosslinking, require lengthy and numerous amplifications, and sometimes carrier DNA, all before preparing the library.
This team ditched the cross-linking and optimized a Native ChIP protocol.
Native ChIP (N-ChIP) has higher resolution than cross-linking ChIP (X-ChIP), and doesn’t have the non-specific interactions that sometimes accompany cross-linking. Historically, N-ChIP has been primarily used when cross-linking wasn’t required (e.g. tightly bound proteins like histones etc.,), but it has been applied to scoping out some of more loosely interacting transcription factors as well.
The researchers’ new protocol is based on works with 200-times fewer cells than a previously reported N-ChIP method and didn’t sacrifice much on performance metrics.
- Sensitivity was good down to 100,000 cells per IP, but it did decrease with decreasing cell numbers, down to 20,000 cells per IP.
- The method was specific, with more than 90% of the peaks matching up with those of the previously reported method.
- Also, the specificity remained the same, even when using very few cells.
After initial tests of the method, the researchers successfully analyzed real-world samples of lymphocytes from human twins. (The samples were in the range of 365-500,000 cells for each IP.)
See how low they could go at BMC Genomics, November 2012