EpiNext cTIP (CUT&Tag In-Place)-Sequencing Kit

The innovative working principle, optimized protocol, and components of the EpiNext™ cTIP (CUT&Tag In-Place)-Sequencing Kit allow for the capture of targeted protein/DNA complexes with minimized non-specific background levels and for the construction of both non-barcoded (singleplexed) and barcoded (multiplexed) libraries quickly in order to map target protein-DNA interaction regions with less bias and at a high resolution

Product Description

The EpiNext™ cTIP (CUT&Tag In-Place)-Sequencing Kit is a complete set of reagents required for carrying out a successful cTIP-Seq starting from mammalian cells.

The kit is designed to enrich a protein (histone or strong binding transcription factor)- specific DNA complex from low input cells/chromatin and to prepare a library for next-generation sequencing using Illumina platforms such as Illumina Genome Analyzer II, HiSeq, and MiSeq systems. 

Features

• High enrichment
• Low input materials
• In place tagging
• Minimal background
• Fast, streamlined procedure
• Highly convenient

Background Information

Enrichment of histone or transcription factor (TF)-complexed DNA in vivo followed by next-generation sequencing offers an advantageous tool for studying genome-wide protein-DNA interactions. It allows for the analysis of a specific protein binding to DNA sequences in living cells. Such analysis requires that the method reliably identify true target protein-enriched regions, particularly from limited cell samples.

These samples could include rare cell populations isolated from tissues, specific cells sorted from entire cell populations, and primary cultured sub-population cells such as embryonic cells. In addition, the method should ensure that enriched DNA contains minimal background and that the mapping of protein-DNA binding regions is with minimal bias and at high resolution.

A major method used for a long time to achieve this goal is chromatin immunoprecipitation followed by sequencing (ChIP-Seq). However, the major limitation for ChIP is that it needs

• 1) a large amount of input material, cells or tissue, to produce a strong enough signal over background noise; and
• 2) cross-linking during an initial fixation step.

There are several advanced methods available for ChIP-Seq with reduced cell numbers or increased resolution. These methods include ChIP-exo, ChIPmentation. ChIP-exo provides high-resolution mapping, but is time-consuming and requires an ample supply of input cells.

While ChIPmentation uses transposase and sequencing-compatible adaptors to enable the integration of ligation during the ChIP process, it follows a traditionally slow (2 day) ChIP procedure and cannot achieve high-resolution mapping.

Fig 1. Workflow of theEpiNext™ cTIP (CUT&Tag In-Place)-Sequencing Kit

Principle & Procedure

The kit contains all necessary reagents required for carrying out a successful cTIP-Seq starting from mammalian cells. In the reaction, nuclei is isolated from cells. The target protein-DNA complex is bound/captured with the CHIP-grade antibody of interest. With the use of a unique nucleic acid cleavage enzyme mix, chromatin is fragmented, and DNA sequences in both ends of the target protein/DNA complex are cleaved/removed. All the while, the DNA sequence occupied by the target protein is unaffected.

The adaptors are ligated to the target protein-bound DNA fragments on the beads. The ligated DNA is then released, purified, and amplified with a high-fidelity PCR mix for library DNA construction. DNA is then cleaned, released, and eluted.

Included in the kit are a positive control antibody (anti-H3K9me3), a negative control non-immune IgG, and control chromatin, which can be used to demonstrate the efficacy of the kit and performance at the PCR/bioanalyzer step.

Fig2. Size distributionof library fragmentsusingtheEpiNext™ cTIP (CUT&Tag In-Place)-Sequencing Kit:Histone/DNAcomplexwas capturedfrom 500ng of chromatin isolated from Hela cells bythe control antibody (H3K9me3)andused for DNA library preparation.Peak at 295 bps reflects theinsert size of mononucleosome(around 150 bps)

Fig3. Size distributionof library fragmentsusingtheEpiNext™ cTIP (CUT&Tag In-Place)-Sequencing Kit:RNA polymerase II/DNAcomplexwas enrichedfrom 50,000 Jurkat cells byanti-RNA polymerase II (PoLII) antibodyandused for DNA library preparation.Peak at 280 bps reflectsinsert size of PoLII occupancy(around 130 bps).