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TECH NOTE

Ligation-Free ChIP-Seq Library Preparation

The DNA SMART ChIP-Seq Kit and ChIP Elute Kit

Overview

Pushing good science forward sometimes demands working through experimental challenges. Preparing next-generation sequencing (NGS) libraries from chromatin immunoprecipitation (ChIP) experiments can be one of those challenges due to the small amount of DNA available and the time required to prepare samples for sequencing. Most library preparation methods rely on ligation to add sequencing adapters when generating ChIP sequencing (ChIP-seq) libraries. However, these methods require double-stranded DNA (dsDNA) inputs, limiting which methods can be used to elute DNA from beads during ChIP or requiring additional preparation steps prior to library construction. The ChIP Elute Kit employs a simple and fast method for cross-linking reversal and eluting ChIP DNA. It generates single-stranded DNA (ssDNA) directly compatible with the DNA SMART ChIP-Seq Kit, which utilizes a modified version of SMART template switching technology to provide a ligation-free method for addition of Illumina® sequencing adapters. Template switching technology allows for single-step adapter addition, and has the sensitivity required for library preparation from picogram quantities of nucleic acids.

The ChIP Elute Kit and DNA SMART ChIP-Seq Kit are robust and reliable tools for ChIP-seq applications, particularly at low input levels (100 pg–10 ng). Using these two kits, high-quality library preparation can be completed in an efficient and time-saving manner.

Template Switching Technology for DNA

DNA SMART technology eliminates the need for an adapter ligation step and associated cleanup during ChIP-seq library preparation. This improves library yield and maintains the complexity in the original DNA sample. The streamlined protocol is enabled by the SMARTScribe Reverse Transcriptase (RT) which copies the DNA template and adds a few additional nucleotides to the 3' end of the newly synthesized DNA. The carefully designed DNA SMART Oligonucleotide base-pairs with these additional non-template nucleotides and creates an extended template, enabling the SMARTScribe RT to continue replicating to the end of the oligonucleotide. Sequencing libraries are then amplified by PCR using primers containing Illumina adapters

DNA SMART ChIP-Seq Kit technology

Flowchart of technology in the DNA SMART ChIP-Seq Kit. This single-tube workflow allows users to generate Illumina-compatible libraries for ChIP-seq experiments. After library size selection and purification, the total time from input DNA to ChIP-seq library is approximately four hours.

Fast ChIP Elution

The ChIP Elute Kit is a fast and simple tool for dissociation of the nuclear protein–DNA complexes (cross-linking reversal) and elution of genomic DNA fragments from ChIP experiments. Recovering purified DNA from ChIP can be a tedious aspect of traditional ChIP protocols and may take up to overnight; however, with the ChIP Elute Kit, purified ChIP DNA can be recovered as ssDNA in approximately one hour. ssDNA is compatible with downstream applications such as qPCR or ChIP-seq library preparation using the DNA SMART ChIP-Seq Kit. The ChIP-seq libraries generated using the ChIP Elute Kit are equivalent to those generated from longer, traditional methods of cross-linking reversal and elution, but in a significantly shorter time.

Sequencing metrics comparing the ChIP Elute Kit to traditional cross-linking reversal methods
Elution/cross-linking reversal method ChIP Elute Traditional ChIP Elute Traditional
Input amount 1 ng 0.25 ng
Library yield (nM) 21.4 16.4 37.3 28.1
Mapped to genome (%) 89.7 90.2 86.4 87.3
Mapped uniquely to genome (%) 71.6 73.2 70.2 71.1
Useful reads (uniquely mapped, non-duplicates; %) 66.0 67.2 58.1 59.0
Non-redundant rate 0.92 0.96 0.83 0.83
No. of peaks identified 37,610 36,924 39,525 37,134

Sequencing metrics comparing ChIP-seq libraries generated with ChIP Elute or traditional cross-linking reversal methods. ChIP was performed using an antibody against CTCF then DNA was eluted using the two different methods. 1 ng or 0.25 ng of the resulting DNA was then used to generate ChIP-seq libraries with the DNA SMART ChIP-Seq Kit using 13 PCR cycles for the 1 ng libraries and 16 PCR cycles for the 0.25 ng libraries. Across all sequencing metrics, ChIP DNA produced using the ChIP Elute Kit generated ChIP-seq libraries that were comparable to those produced using traditional, slow cross-linking reversal methods. Showing that the ChIP Elute Kit works well across a wide range of inputs in just one hour.

The peaks identified from libraries made from DNA eluted with either the ChIP Elute Kit or the traditional method are very similar and match those identified by the ENCODE project. The number of peaks identified is consistent across input levels for both the ChIP Elute Kit and traditional method, and the number of peaks identified by either method is similar for the same input level.

DNA SMART ChIP-Seq Kit technology

The shape and location of peaks are similar for different cross-linking reversal and elution methods and across input amounts. ChIP-seq libraries made from DNA eluted with either the ChIP Elute Kit or traditional method had high amounts of overlap in the number of peaks identified. The percent overlap of peaks identified in ChIP-seq libraries prepared from DNA isolated with the ChIP Elute and traditional method were 90% and 89% for 1 ng and 250 pg samples, respectively.

Protocol Improvements for ChIP-Seq Library Preparation

Contrary to typical library preparation protocols with an absolute requirement for a clean-up step between adapter ligation and PCR amplification of the library, the DNA SMART ChIP-Seq Kit uses a combined size selection and clean-up step only after library amplification by PCR. The post-PCR size selection results in a higher yield and better library complexity (non-redundant rate) while maintaining the quality of the libraries.

Sequencing metrics comparing pre- and post-PCR size selection
Size selection pre-PCR post-PCR post-PCR (single selection)
Library yield (nM) 10.9 17.2 26.9
No. uniquely mapped reads (million) 5.1 5.9 4.3
Non-redundant rate 0.75 0.85 0.89
Peaks identified with 3.85 M reads (uniquely mapped, non-duplicates) 32,827 34,011 33,398
DNA SMART ChIP-Seq Kit technology

Sequencing metrics from ChIP-seq libraries purified before or after amplification. ChIP was performed using an antibody against CTCF, then DNA was eluted using the traditional method. ChIP-seq libraries were generated from 200 pg of the ChIP DNA with size selection before or after library amplification (16 cycles of PCR). Size selection removed both small and large inserts (except for the single size selection sample indicated, which only removed small fragments/adapter dimers).

Performing size selection after amplification makes the workflow easier while generating data comparable to those generated using a pre-PCR size selection. The location and shape of the peaks identified using post-PCR size selection are indistinguishable from those reported by the ENCODE project.

Peak calling and electropherograms of pre- and post-PCR size selection libraries

Size selection before or after PCR amplification does not affect library quality. Peaks identified from pre- or post-PCR size selection and purification (Panel A) and electropherograms showing the different libraries generated using pre- or post-PCR size selection (Panel B) are shown.

Sensitive Library Production

The DNA SMART ChIP-Seq Kit has the sensitivity to generate sequencing libraries from very small amounts of fragmented ssDNA or dsDNA. The number of unique, non-duplicate reads is high across all input levels, and the number of peaks identified is similar across input amounts.

Sequencing metrics from various amounts of input DNA
Input amount (ng) 4 1 0.5 0.25 0.1 0.05
No. of PCR cycles 12 13 14 15 17 18
Library yield (nM) 44.5 19.2 32.2 12.0 24.3 14.3
Mapped to genome (%) 91.9 91.0 91.0 91.1 90.7 90.2
Mapped uniquely to genome (%) 72.8 72.5 72.3 72.7 72.1 71.8
Useful reads (uniquely mapped, non-duplicates; %) 68.2 64.4 64.3 50.3 45.0 23.8
No. of peaks identified 16,738 16,811 16,366 17,277 16,584 19,601

Sequencing metrics from specific amounts of input DNA. ChIP was performed using an anti-H3K4me3 antibody and DNA was eluted using the traditional method. ChIP-seq libraries were then generated from different dilutions of the same dsDNA ChIP input sample. These libraries have very similar sequencing metrics, showing the high sensitivity and reproducibility of this kit.

Libraries generated with this kit have high reproducibility; the overlap between the number of peaks identified from technical replicates generated from the same amount of input DNA is >93% for input levels greater than 100 pg, and libraries generated from different input amounts have >94% overlap with each other. The complexity (the non-redundant rate) is also very high for these libraries.

ChIP-seq library complexity and reproducibility from low-input DNA.

ChIP-seq library complexity and reproducibility is maintained across input amounts. The reproducibility between technical replicates was similar across input amounts (Panel A). The non-redundant rate (normalized for 10 million uniquely mapped reads) was well above the standard recommended by the ENCODE project (0.8) for inputs >0.5 ng (Panel B; error bars indicate the standard deviation of two technical replicates). Compared to the 4 ng library, the number of peaks identified was similar across lower input libraries (Panel C). The shape and location of the peaks was similar across input levels, and matched very well to ENCODE data (293 cells, anti-H3K4me3 antibody, U. Washington), even for as little as 50 pg input DNA (Panel D).

Reproducible Libraries from Specific Numbers of Cells

Exact quantification of DNA obtained by ChIP can be very difficult due to the low concentrations. Typically, researchers must use the entire ChIP DNA sample obtained for sequencing library preparation. With DNA SMART technology, ChIP-seq data from total (unquantified) ChIP DNA is consistent across different starting cell numbers.

Sequencing metrics from total DNA from specified numbers of cells
Input amount (millions of cells) 1 0.2 0.05 0.01
No. of PCR cycles 15 18 18 18
Library yield (nM) 86.7 101 44.6 20.5
Mapped to genome (%) 92.7 88.6 84.3 75.8
Mapped uniquely to genome (%) 79.0 74.8 70.4 59.6
Useful reads (uniquely mapped, non-duplicates; %) 66.8 63.5 49.9 34.0
Non-redundant rate 0.85 0.85 0.71 0.57
No. of peaks identified 19,459 19,339 18,549 22,564

The DNA SMART ChIP-Seq Kit generates high-quality libraries from low cell number ChIP experiments. Total ssDNA recovered using the ChIP Elute Kit from ChIP experiments using an anti-H3K4me3 antibody with the number of cells indicated was used as input for the DNA SMART ChIP-Seq Kit. Mapping statistics were very good across all input levels.

DNA SMART ChIP-Seq Kit libraries maintain consistent representation of sequences even at the lowest input levels. Between 86–91% of the identified peaks overlapped between different input amounts. Across all cell inputs, the DNA SMART ChIP-Seq Kit generates data that matches well with the reported data from the ENCODE project (>87 % overlap in peaks).

ChIP-seq library complexity and reproducibility from total DNA from small Amounts of cells; DNA SMART ChIP-seq library overlap with ENCODE data

Peak overlap between libraries generated from various inputs. Peaks from different numbers of cells were identified using 6 million (for 10,000 cells) or 9–10 million (for 50,000–1,000,000 cells) uniquely mapped reads. There was a high amount of overlap across the number of input cells (Panel A). The peaks were of similar shape across cell inputs and matched the peaks obtained by the ENCODE project (293 cells, anti-H3K4me3 antibody, U. Washington; Panel B). Additionally, the total number of peaks overlapping with those identified in the ENCODE project was high (Panel C).

Summary

The modified template switching technology at the core of the DNA SMART ChIP-Seq Kit provides a sensitive means for generating sequencing libraries in a ligation-independent manner from either ssDNA or dsDNA templates. The combined post-PCR library size selection and clean-up step generates libraries with higher yields than if size selection and cleanup are performed before library amplification, thus simplifying the workflow, and making this kit ideal for low-input DNA samples. The ChIP Elute Kit minimizes the time needed to reverse cross-link, elute, purify, and concentrate ChIP DNA. This makes it faster to get to downstream applications, like ChIP-seq. Together the ChIP Elute Kit and DNA SMART ChIP-Seq Kit generate sensitive, robust, reproducible ChIP-seq libraries for Illumina sequencing.

Methods and References

For ChIP assays, HEK 293T cells were grown to 80% confluence and fixed with 1% formaldehyde for 10 minutes. ChIP was performed with ChIP-grade anti-H3K4me3 or anti-CTCF antibodies according to standard methods (chromatin shearing by sonication with Bioruptor Pico; Diagenode). After washes, DNA–protein complexes immobilized to Protein A/G agarose beads (Santa Cruz Biotechnologies, Inc.) were processed according to the traditional method (see below) or the ChIP Elute Kit.

Traditional ChIP DNA recovery method: DNA-protein complexes were eluted from the beads in SDS buffer (1% SDS, 0.1 M NaHCO3) then DNA–protein cross-links were reversed in the presence of 250 mM NaCl at 65°C overnight. Finally, a Proteinase K treatment was performed to digest proteins. See Massie and Mills (2012) for an example of this method. After elution and cross-linking reversal, the DNA was purified and concentrated with a Macherey-Nagel NucleoSpin Gel and PCR Clean-Up kit (using the NTB buffer; available separately).

Samples processed with ChIP Elute Kit were handled according to the kit protocol. The ChIP Elute Kit includes DNA clean-up and concentration as part of the protocol, additional purification prior to sequencing library preparation is not necessary.

For direct comparison between the ChIP Elute Kit and the traditional method, beads with bound complexes were split after the last wash step, and half of the material was processed with the traditional method, while the other half was processed with the ChIP Elute Kit.

Sequencing libraries were generated using the DNA SMART ChIP-Seq Kit and size selection was performed with AMPure XP beads (Beckman Coulter) using Option 1 or Option 4 as described in the DNA SMART ChIP-Seq Kit User Manual. Sequencing was carried out on Illumina MiSeq® or HiSeq® 2500 instruments. All runs were paired-end sequenced using the Custom Read2 Seq Primer from the DNA SMART ChIP-Seq Kit for some runs.

Mapping of reads (unpaired) to the human genome (hg19) was performed using Bowtie2 with default settings (plus trimming of the first three 5' nucleotides of the reads obtained with the Read Primer 1). Uniquely mapping reads were selected and the SAM files were sorted and converted to BAM files using SAMTOOLS. Peaks were identified using MACS version 1.4 (default settings except the p-value cutoff set at 1 x 10-7). Raw data generated by the ENCODE consortium were downloaded as fastq files from http://genome.ucsc.edu/ENCODE/downloads.html and analyzed similarly to the data generated with the DNA SMART ChIP-Seq Kit. Reads and peaks were visualized using the UCSC genome browser.

References:

Massie, C., and Mills, I. (2012) In Transcriptional Regulation: Methods and Protocols, Ed. Vancura, A. (Springer Press, NY) pp. 157–173.

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