Limits...
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: ChIP-seq is the method of choice for genome-wide studies of protein-DNA interactions.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.


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

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

Fig1: 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.

Mentions: We reasoned that when the desired end product of a ChIP experiment is a sequencing library, it would be advantageous to perform library reactions during the ChIP step rather than after DNA purification. This makes the protocol faster and easier to carry out, since the need for precipitations or column purifications between the reactions is removed. We performed ChIP using standard protocols until the IP washes and end repair, A-tailing and ligation reactions were done directly on cross-linked chromatin attached to magnetic beads (Figure 1a), with brief washes with PBS in between the reactions to remove enzymes. Besides reducing the risk of sample cross-contamination, the ligation of barcoded adapters before elution means that most adaptor dimers will be in solution and can easily be removed to allow amplification of the sample without a prior bead- or gel-based size selection. In comparison, standard ChIP-seq protocols followed by the Illumina TruSeq ChIP-seq library protocol takes 4–5 days even with recent modifications to remove several laborious spin-column steps (Additional file 1: Figure S1). The cost per reaction for lobChIP is lowered by more than tenfold compared to TruSeq and is also lower than other protocols using off-the-shelf reagents, since less purification reagents are needed.Figure 1


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)

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

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

Fig1: 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.
Mentions: We reasoned that when the desired end product of a ChIP experiment is a sequencing library, it would be advantageous to perform library reactions during the ChIP step rather than after DNA purification. This makes the protocol faster and easier to carry out, since the need for precipitations or column purifications between the reactions is removed. We performed ChIP using standard protocols until the IP washes and end repair, A-tailing and ligation reactions were done directly on cross-linked chromatin attached to magnetic beads (Figure 1a), with brief washes with PBS in between the reactions to remove enzymes. Besides reducing the risk of sample cross-contamination, the ligation of barcoded adapters before elution means that most adaptor dimers will be in solution and can easily be removed to allow amplification of the sample without a prior bead- or gel-based size selection. In comparison, standard ChIP-seq protocols followed by the Illumina TruSeq ChIP-seq library protocol takes 4–5 days even with recent modifications to remove several laborious spin-column steps (Additional file 1: Figure S1). The cost per reaction for lobChIP is lowered by more than tenfold compared to TruSeq and is also lower than other protocols using off-the-shelf reagents, since less purification reagents are needed.Figure 1

Bottom Line: ChIP-seq is the method of choice for genome-wide studies of protein-DNA interactions.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.