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染色质免疫共沉淀技术（Chromatin Immunoprecipitation，ChIP）也称结合位点分析法，是研究体内蛋白质与DNA相互作用的有力工具，通常用于转录因子结合位点或组蛋白特异性修饰位点的研究。将ChIP与第二代测序技术相结合的ChIP-Seq技术，能够高效地在全基因组范围内检测与组蛋白、转录因子等互作的DNA区段。

ChIP-Seq的原理是：首先通过染色质免疫共沉淀技术（ChIP）特异性地富集目的蛋白结合的DNA片段，并对其进行纯化与文库构建；然后对富集得到的DNA片段进行高通量测序。研究人员通过将获得的数百万条序列标签精确定位到基因组上，从而获得全基因组范围内与组蛋白、转录因子等互作的DNA区段信息。

 

   At Boston University, the Galagan lab is trying to get a complete picture of Mycobacterium tuberculosis — in particular, how the organism’s regulatory network functions. By methodically performing ChIP-seq on each transcription factor in the microbe, the scientists are using sequence data to determine which other transcription factors and genomic regions are expressed as a result. The ultimate goal: a comprehensive view of the complex interactions among these transcription factors across the whole TB genome.

The scientist behind the lab’s library prep is Chris Mawhinney, who says that using Pippin Prep for size selection in the ChIP workflow saves her time and eliminates the possibility of sample cross-contamination. Mawhinney performs size selection after adapter ligation and purification as she preps libraries for sequencing on the Illumina GAIIx. “If you have too broad a range of sizes, the sequencer software has issues locating the clusters,” she says. “And adapter-dimers will anneal to your flow cell and eat up your reads, wasting capacity.”

Mawhinney uses Pippin to select for 250 base pairs, which allows her to remove the low molecular weight content as well as larger fragments. “With Pippin Prep, you can get it down to a really nice, narrow size,” she says, noting that the tool is easy to set up and run.

In addition to the time savings of using an automated solution instead of a manual gel, Mawhinney says Pippin speeds up the sample prep routine because there’s no cleanup needed. “The great thing about Pippin is that you can go right from size selection to PCR; there’s no middle cleanup step, so it’s very convenient,” she adds. “That’s a huge plus and it saves a lot of time.”

Preventing contamination is also critical in a lab where samples are often used up completely during sequencing. Mawhinney treats each sample as precious, so she can’t afford to lose anything to contamination or to poor sizing. On Pippin, she says, “the channels are all separate, so that gets rid of the possibility of contamination.”

For more on the Galagan lab’s interrogation of transcription factors in TB, check out their recent Nature paper.