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

Wallerman O, Nord H, Bysani M, Borghini L, Wadelius C - Epigenetics Chromatin (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, BMC, Uppsala University, Box 815, 75108 Uppsala, Sweden ; Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.

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.

No MeSH data available.


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).
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Fig3: 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).

Mentions: One of the main objectives with the lobChIP protocol was to simplify the handling of multiple samples to allow more factors to be studied in parallel. We designed a multiplex experiment where the volumes of chromatin and washings were reduced to 150 µl, which allows the use of standard PCR strips and multichannel pipettes. We have previously studied the liver-specific TFs FOXA1, FOXA2 and HNF4a in HepG2 cells [7, 8], and to continue this work, we repeated these experiments and further included antibodies targeting TCF7L2, HNF6 and NRF1, since motifs for these TFs were found to be overrepresented in proximity to the previously studied factors [7, 9]. We also included antibodies for the more general factors, CTCF and Pol II, as well as for the histone modification H3K36me3 and a negative control (IgG). The protocol was adjusted, so that all steps from chromatin to amplified library could be carried out in a single day (“Methods”; Figure 1a), using a 4-h immunoprecipitation with chromatin from approximately 5 million cells per reaction. After washes and library reactions, each sample was divided into two to further compare the direct and standard elution methods described above by sequencing the two amplified libraries from each ChIP reaction on separate lanes (Additional file 1: Figure S5). We found that directly amplified samples had a slightly higher adaptor dimer contamination, but all samples had more than 90% aligned reads and a low level of PCR duplicates (Additional file 2). Peak finding and de novo motif prediction on combined reads were then used to validate that all lobChIP experiments for the sequence-specific TFs had been successful. The peak lists were further validated by comparison to ENCODE results. As can be expected, the ENCODE datasets which were produced from individual experiments using a larger number of cells and deeper sequencing had a larger number of peaks for all factors except NRF1, but there was a good agreement among the called peaks with overlaps as high as 86–99% for the smaller datasets (Figure 3a). Overall, this experiment shows that a single-day lobChIP protocol works well for both TFs and histone modifications, and further that potential tissue-specific regulatory elements can be identified using a combination of related TFs, as exemplified with the intragenic region of TBC1D4 where binding sites for four liver-specific TFs were identified (Figure 3b).Figure 3


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

Wallerman O, Nord H, Bysani M, Borghini L, Wadelius C - Epigenetics Chromatin (2015)

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).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4507313&req=5

Fig3: 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).
Mentions: One of the main objectives with the lobChIP protocol was to simplify the handling of multiple samples to allow more factors to be studied in parallel. We designed a multiplex experiment where the volumes of chromatin and washings were reduced to 150 µl, which allows the use of standard PCR strips and multichannel pipettes. We have previously studied the liver-specific TFs FOXA1, FOXA2 and HNF4a in HepG2 cells [7, 8], and to continue this work, we repeated these experiments and further included antibodies targeting TCF7L2, HNF6 and NRF1, since motifs for these TFs were found to be overrepresented in proximity to the previously studied factors [7, 9]. We also included antibodies for the more general factors, CTCF and Pol II, as well as for the histone modification H3K36me3 and a negative control (IgG). The protocol was adjusted, so that all steps from chromatin to amplified library could be carried out in a single day (“Methods”; Figure 1a), using a 4-h immunoprecipitation with chromatin from approximately 5 million cells per reaction. After washes and library reactions, each sample was divided into two to further compare the direct and standard elution methods described above by sequencing the two amplified libraries from each ChIP reaction on separate lanes (Additional file 1: Figure S5). We found that directly amplified samples had a slightly higher adaptor dimer contamination, but all samples had more than 90% aligned reads and a low level of PCR duplicates (Additional file 2). Peak finding and de novo motif prediction on combined reads were then used to validate that all lobChIP experiments for the sequence-specific TFs had been successful. The peak lists were further validated by comparison to ENCODE results. As can be expected, the ENCODE datasets which were produced from individual experiments using a larger number of cells and deeper sequencing had a larger number of peaks for all factors except NRF1, but there was a good agreement among the called peaks with overlaps as high as 86–99% for the smaller datasets (Figure 3a). Overall, this experiment shows that a single-day lobChIP protocol works well for both TFs and histone modifications, and further that potential tissue-specific regulatory elements can be identified using a combination of related TFs, as exemplified with the intragenic region of TBC1D4 where binding sites for four liver-specific TFs were identified (Figure 3b).Figure 3

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, BMC, Uppsala University, Box 815, 75108 Uppsala, Sweden ; Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.

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.

No MeSH data available.