Chromatin immunoprecipitation sequencing (ChIP-seq) is an invaluable tool for interrogating genomic loci that are targeted by DNA-binding proteins of interest. One particularly useful application is identifying the binding sites of transcription factors, which are responsible for the determination of cell fate and play a key role in development. However, as transcription factors involved in cell fate may only be expressed in specific or rare cell type(s), ChIP-seq experiments can be challenging due to highly limited samples.

To address this difficulty, Dr. Yuri Yasuoka (RIKEN Biomedical Science Research Center) used our ThruPLEX DNA-Seq Kit to generate sequencing libraries for efficient ChIP-seq analysis of transcription factors from ~250 Xenopus gastrulae.

The researchers generated ThruPLEX sequencing libraries from ~250 Xenopus gastrulae using either total ("Input") or Otx2-immunoprecipitated (anti-Ox2; "Otx2 ChIP") DNA. ThruPLEX libraries were sequenced on an Illumina NextSeq® 550 instrument with 75-bp paired-end reads and mapped to the Xenopus genome using the Burrows-Wheeler aligner. Sequencing metrics demonstrated comparable total reads, mapped reads, and mapped read percentages between Input and Otx2 ChIP samples (Table I).

Mapped reads from Otx2 ChIP samples corresponding to regions of interest were visualized with the Integrative Genomics Viewer (IGV) (Figure 1). Sequencing mapping results were concordant with a prior ChIP-seq study (Yasuoka et al. 2014). Notably, this prior study used a number of Xenopus gastrulae (~2,000–3,000) that is an order of magnitude greater than the present study (~250 gastrulae). The researchers also noted that a sufficient amount of library was able to be generated from ~1 ng of DNA (12 PCR cycles in the ThruPLEX protocol), speaking to the exquisite sensitivity of our ThruPLEX DNA-Seq Kit.

While ChIP-seq is an extremely powerful tool for querying the binding domain(s) of DNA-binding proteins, sample input requirements can present significant hurdles, particularly for rare targets. In the present study, our ThruPLEX chemistry was used to generate ChIP-seq libraries from ~250 Xenopus gastrulae, representing a ~10-fold improvement over a prior study that did not use our ThruPLEX chemistry (Yasuoka et al. 2014). Based on yield and sensitivity, the researchers noted that, depending on the specific antibody and its target, it may be feasible to use inputs of as little as 50 gastrulae. These results demonstrate the ability of our ThruPLEX DNA-Seq Kit to generate DNA-seq libraries from extremely limited samples and present a highly sensitive method of querying rare targets in ChIP-seq.

Chromatin extraction and shearing

Approximately 250 Xenopus gastrulae were fixed with 1% formalin and stored at –80°C until processing. Fixed gastrulae were then lysed with lysis buffer, nuclei were concentrated, and chromatin was fragmented with a Picorupter sonication device (Diagenode). A fraction of fragmented chromatin was saved for the Input sample.

Chromatin immunoprecipitation and DNA purification

An anti-Otx2 antibody was conjugated to Protein A magnetic beads, antibody-bound beads were added to the fragmented chromatin, and the mixture was allowed to incubate overnight at 4°C with rotation. Beads were then washed once with RIPA buffer and incubated at 65°C overnight in elution buffer containing 1% SDS. Following RNase A treatment and decrosslinking with Proteinase K, DNA was purified with a MinElute PCR Purification Kit (QIAGEN) using two elutions of 10 µl each. Recovered DNA was quantified using a dsDNA HS kit (Qubit).

Library preparation and sequencing

DNA-seq libraries were prepared with the ThruPLEX DNA-Seq Kit using 10 µl of either Otx2 ChIP or Input DNA. ThruPLEX libraries were sequenced on an Illumina NextSeq 550 with 75-bp paired-end reads.

Data analysis

Sequencing reads were mapped to the Xenopus genome (v9) using the Burrows-Wheeler aligner, and genomic regions were visualized with the Integrative Genomics Viewer (Broad Institute).

Yasuoka, Y. et al. Occupancy of tissue-specific cis-regulatory modules by Otx2 and TLE/Groucho for embryonic head specification. Nat. Commun. 5, 4322 (2014).