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. 2015 Jul 21:8:25.
doi: 10.1186/s13072-015-0017-5. eCollection 2015.

lobChIP: from cells to sequencing ready ChIP libraries in a single day

Ola Wallerman  1 , Helena Nord  2 , Madhusudhan Bysani  3 , Lisa Borghini  4 , Claes Wadelius  2
Affiliations

lobChIP: from cells to sequencing ready ChIP libraries in a single day

Ola Wallerman et al. Epigenetics Chromatin. .

Abstract

Background: ChIP-seq is the method of choice for genome-wide studies of protein-DNA interactions. We describe a new method for ChIP-seq sample preparation, termed lobChIP, where the library reactions are performed on cross-linked ChIP fragments captured on beads.

Results: The lobChIP method was found both to reduce time and cost and to simplify the processing of many samples in parallel. lobChIP has an early incorporation of barcoded sequencing adaptors that minimizes the risk of sample cross-contamination and can lead to reduced amount of adaptor dimers in the sequencing libraries, while allowing for direct decross-linking and amplification of the sample.

Conclusions: With results for histone modifications and transcription factors, we show that lobChIP performs equal to or better than standard protocols and that it makes it possible to go from cells to sequencing ready libraries within a single day.

Keywords: ChIP-seq; Chromatin immunoprecipitation; Illumina; Library preparation; NGS.

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Figures

Figure 1
Figure 1
a Flowchart of the lobChIP procedure with timings used in the 1-day protocol. b–e Comparison of lobChIP to ENCODE data for histone modifications. b Clustering of read intensities at TSS for four different histone modifications from ENCODE (-E) and lobChIP. c PCA plot of the four different histone modifications, with ENCODE in dark colors and lobChIP in light colors. d Representative enrichment for H3K36me3, H3K4me3 and H3K27me3 over a 1 Mb window of chromosome 11. Corresponding ENCODE results are given as dense tracks above the RefSeq genes. e Scatter plot comparing lobChIP with ENCODE read counts for H3K27me3 in 40 kb windows centered at TSS.
Figure 2
Figure 2
Comparison of direct elution and amplification to SDS elution followed by decross-linking and purification for H3K27ac. a Representative signals of enrichment for a TSS (left) and distal site (right). b Scatter plot of reads at TSS for the two different elutions and amplifications from the same lobChIP sample. c Comparison of enrichment at TSS for the directly eluted lobChIP sample and a sample done with standard ChIP-seq protocol. Read counts on x- and y-axis are normalized to sequencing depth.
Figure 3
Figure 3
Results from manual and automated multiplexed lobChIP runs. a Motifs identified de novo for seven TFs from the manual 1-day lobChIP experiment, with the number of identified peaks and percentage of peaks overlapping (within 1 kb) with ENCODE peaks. (Asterisk) For NRF1 where more peaks were identified in our dataset, the percentage of ENCODE overlapping with our peaks is given. b Normalized signals (RPM) for H3K36me3, Pol II and TFs over the TBC1D4 gene with an enlarged region illustrating motif positions and peaks for FOXA1/2, TCF7L2 and HNF6 at distinct locations. c Automated lobChIP results for FOXA1 give an enrichment profile similar to the ENCODE sample, as exemplified here for the APOA5–APOA1 region. d Venn diagram for FOXA1 overlaps for automated lobChIP (blue) and standard ChIP-seq protocols on the SOLiD instrument [8] (red). e Venn diagram of overlaps between genes with enrichment for H3K4me3 in the AHT-ChIP-seq study (blue) and in the automated lobChIP run (green).
Figure 4
Figure 4
Relplicate analysis. a FOXA2 read counts at peak locations for two technical replicates. b Scatter plot comparing reads at TSS for two lobChIP technical replicates for H3K4me3. c H3K27ac results for automated and manual lobChIP at TSS. Read counts on x- and y-axis are normalized to sequencing depth. d Five replicates for NRF1, with replicate 2–4 (blue bar) from the same chromatin preparation. The number of peaks is given on the diagonal with correlation coefficients for enrichment below. e Four-way venn diagram for genes with NRF1 peaks, with merged results for the three NRF1 samples made from the same chromatin. f Overlap of TCF7L2 peaks identified in ENCODE, SOLiD lobChIP and standard Illumina ChIP-seq.
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References

    1. Kilpinen H, Waszak SM, Gschwind AR, Raghav SK, Witwicki RM, Orioli A, et al. Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription. Science. 2013;342(6159):744–747. doi: 10.1126/science.1242463. - DOI - PMC - PubMed
    1. Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-wide mapping of in vivo protein-DNA interactions. Science. 2007;316(5830):1497–1502. doi: 10.1126/science.1141319. - DOI - PubMed
    1. O’Geen H, Echipare L, Farnham PJ. Using ChIP-seq technology to generate high-resolution profiles of histone modifications. Methods Mol Biol. 2011;791:265–286. doi: 10.1007/978-1-61779-316-5_20. - DOI - PMC - PubMed
    1. ENCODE Project Consortium An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489(7414):57–74. doi: 10.1038/nature11247. - DOI - PMC - PubMed
    1. Solomon MJ, Varshavsky A. Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc Natl Acad Sci U S A. 1985;82(19):6470–6474. doi: 10.1073/pnas.82.19.6470. - DOI - PMC - PubMed