The TruSeq ChIP Sample Prep kit generates Paired End (PE), indexed libraries that are compatible with all Illumina sequencing platforms and can be multiplexed. The legacy ChIP-Seq Sample Prep kit generates Single Read (SR) and non-indexed libraries that are compatible with Genome Analyzer, HiSeq and HiScanSQ, but not MiSeq.
This kit is available in a Set A and a Set B, each containing 12 indexes. When used together, sets A and B provide a total of 24 unique indexes.
For some library preparations, AMPure XP beads are user-supplied from Beckman-Coulter Genomics. See the appropriate TruSeq DNA or RNA library prep guide for more information.
Illumina recommends a DNA insert size of 200–800 bp.
Use fluorometric-based methods for quantification, including Qubit or PicoGreen, to provide accurate quantification of ChIP DNA.
UV spectrometer-based methods, such as Nanodrop, measure any nucleotides present in the sample, including RNA, dsDNA, ssDNA, and free nucleotides, which can inaccurately measure the DNA.
ChIP DNA samples can be difficult to fragment, and the wider distribution of starting fragment size is chosen to allow for ease of use with a broader range of starting material. Gel-based size selection focuses on a narrow insert size (250–300 bp post-ligation) to allow for increased success with motif finding, which is improved with tighter distributions and smaller fragments. Illumina recommends using smaller starting fragment sizes, if possible, to increase the amount of material in the selected range. Samples with distributions in the 200–800 bp range have been shown to generate successful libraries, even if the majority of the input material is not in the final size selected.
The protocol is optimized for 18 cycles of PCR to allow for variability in sample input amounts and quality. High quality samples will likely be able to use a lower number of PCR cycles and still generated adequate amounts of final library. A titration of PCR cycles can be performed to identify the optimal performance for particular samples. Illumina does not recommending increasing the number of PCR cycles as it may skew the representation of the library.
The final library shows a size distribution of ~250–300 bp.
Illumina uses a green laser to sequence G/T bases and a red laser to sequence A/C bases. At each cycle at least one of two nucleotides for each color channel need to be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise index read sequencing could fail due to registration failure. For pooling strategies for a small number of samples, please refer to the Adapter Tube Pooling Guidelines section in the library prep guide for the kit you are using. Additionally it is recommended to create a sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations not listed.
Each TruSeq ChIP Library Preparation Kit contains enough reagents and adapters to process 48 samples. Set A and Set B each contain 12 unique indexes, with enough of each index sufficient for 8 individual samples.
The Illumina Experiment Manager (IEM) will notify you if improper combinations are used when creating a sample sheet for use with CASAVA, so it is highly recommended to create your sample sheet prior to performing sample prep/pooling. There are also low plexity pooling guidelines in the Nextera, TruSeq DNA, and TruSeq RNA sample preparation guides. Always pool samples with valid index combinations to avoid image registration failures.
The protocol is optimized for the inclusion of a gel size-selection step selecting for a DNA insert of 150–200 bp. Longer inserts could be used; however, this may decrease the signal to noise for the binding motif and may require modification for optimal run performance. Illumina recommends selecting a gel size range of 250–300 bp for the final library.
Illumina recommends performing the gel size selection step as it is designed to remove unligated adapters, as well as any adapters that might have ligated to one another, and selects a narrow 250–300 bp size-range of DNA fragments for ChIP library construction appropriate for cluster generation.
Longer drying times may be required depending on environmental variables and the amount of ethanol remaining in the well. However, take care not to over-dry beads as this can impact sample recovery.
Illumina’s proprietary method ensures ligation of 2 different adapters in the required orientation to opposing ends of a DNA fragment. PCR selects for these and finalizes the construct ready for hybridizing onto the flow cell surface. Adapter sequences can be determined by sequencing the ligation fragments, but sequence information alone is not sufficient to uncover the method.
The TruSeq ChIP Library Prep Kit does not contain in-line controls due to the low input amount.
Illumina does not provide any recommendations as methods for ChIP pulldown and fragmentation are dependent upon individual antibodies and procedures. Please reference literature or other sources for recommendations.
Illumina recommends using qPCR to quantify TruSeq ChIP libraries. For more information, see Sequencing Library qPCR Quantification Guide.
TruSeq ChIP libraries are compatible with sequencing primers included in all TruSeq cluster kits and in the TruSeq Dual Index Sequencing Primer Box (Single Read or Paired End).
The ChIP-seq workflow generates demultiplexed FASTQ files.
TruSeq ChIP libraries are compatible with all Illumina sequencing platforms, including MiSeq, HiSeq, HiScanSQ and Genome Analyzer.
Sequence TruSeq ChIP libraries on a single-read or paired-end flow cell.
MiSeq runs require a sample sheet at the start of the run to allow analysis. When creating a sample sheet in Illumina Experiment Manager (IEM), you can choose either the FASTQ Only or the ChIP-Seq application in the Other category. Both workflows generate FASTQ files.
The TruSeq ChIP Sample Prep Kit generates paired-end libraries that you can sequence on either single-read or paired-end flow cells. In many cases a single-read (1 x 50 bp) run is sufficient for ChIP-Seq analysis.
Paired-end reads might increase the ability to align reads and resolve reads mapping to repeat sequences. Review the literature and third-party analysis software to determine what type of run is sufficient for the needs of the experiment.
Use CASAVA and MSR for demultiplexing. However, they are not intended for TruSeq ChIP analysis.
There are a number of third party solutions available for the analysis of ChIP data. These include, but are not limited to, MACS, Avadis, and Partek. Please note that Illumina cannot provide support for the use of third party software; please contact the software resources directly with any questions regarding the analysis and use of their software. Additional literature references can be found on the Illumina Epigenetics webpage.