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Jeffrey Kron at
UCONN HEALTH
built this track, and the
Cotney Lab
is responsible for its contents.
Methods Description
All animal work was done in accordance with approved University of Connecticut Health Center IACUC protocols. 4C-seq was performed according to
van de Werken et al. (2012)
with some modifications.
Mouse embryonic tissue was fixed as described in
Cotney et al. (2012)
and nuclei isolated following homogenization with a dounce tissue grinder. Chromatin was digested with the combination NlaIII/DpnII.
Primers were designed using 4cseq primer db from the
Tanay Group
and modified to a split design to permit re-use and multiplexing.
4C-Seq Data Analysis
4C-seq libraries were sequenced using the NextSeq500 (Illumina). Fastq files were demultiplexed by barcode yielding Fastq files for each tissue replicate. Tissue replicate Fastq files were further demultiplexed by viewpoint using Cutadapt
(Martin, 2011).
Trimmed reads were aligned using bowtie2
(Langmead and Salzberg, 2012).
A custom script was used to retain a small percentage of reads 1.5kb upstream or downstream of the viewpoint and normalize the number of reads.
Significant interactions were assessed using r3Cseq
(Thongjuea et al., 2013)
with a modification allowing a larger viewing window near the viewpoint. The significant interactions are represented in the accompanying track hub as a Bed file.
The location of the viewpoint and sequenced interacting fragment are denoted with thick bars. A thin bar is included to clarify the connection between the viewpoint and the distal sites.
All coordinates were then lifted to hg19 using liftOVER
(Kuhn et al., 2013)
Display conventions
Viewpoint Color Legend
- Viewpoint 1 - Viewpoint 1
- Viewpoint 2 - Viewpoint 2
- Viewpoint 3 - Viewpoint 3
- Viewpoint 4 - Viewpoint 4
- Viewpoint 5 - Viewpoint 5
- Viewpoint 6 - Viewpoint 6
References
van de Werken, H.J.G., de Vree, P.J.P., Splinter, E., Holwerda, S.J.B., Klous, P., de Wit, E., and de Laat, W. (2012). 4C Technology: Protocols and Data Analysis. Methods in Enzymology. 2012;513:89-112. doi: 10.1016/B978-0-12-391938-0.00004-5.
Cotney J, Leng J, Oh S, et al. Chromatin state signatures associated with tissue-specific gene expression and enhancer activity in the embryonic limb. Genome Research. 2012;22(6):1069-1080. doi:10.1101/gr.129817.111.
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.Journal. 2011;17, 10-12. doi:http://dx.doi.org/10.14806/ej.17.1.200.
Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature methods. 2012;9(4):357-359. doi:10.1038/nmeth.1923.
Thongjuea S, Stadhouders R, Grosveld FG, Soler E, Lenhard B. r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data. Nucleic Acids Research. 2013;41(13):e132. doi:10.1093/nar/gkt373.
Contacts
Please email Jeffrey Kron or Justin Cotney for questions.
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