Computational protocol: Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line

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Protocol publication

[…] Non-KI(B), KI(B), and KI(MΦ) (2 × 107 each) were subjected to the iChIP procedure as described previously. DT40 was subjected to the in vitro enChIP procedure as described previously. The complex of CRISPR RNA (crRNA) targeting the Pax5 promoter and trans-activating crRNA (tracrRNA) was used as Pax5 gRNA for in vitro enChIP. The gRNA sequences are shown in . Briefly, after fragmentation of chromatin DNA (the average length of fragments was about 2 kbp), the target region was isolated by iChIP or in vitro enChIP. After purification of DNA, DNA libraries were prepared using TruSeq ChIP Sample Prep Kit (Illumina); in this preparation step, DNA fragments around 0.4 kbp in length were selectively concentrated. The libraries were subjected to DNA sequencing using the HiSeq platform according to the manufacturer’s protocol. NGS and data analysis were performed as described previously., Additional information on NGS analysis is provided in . NGS data were mapped onto the reference genome galGal4 using ELAND (Illumina). Narrow peaks of each iChIP-Seq dataset (see Steps 1 and 2 in ) were detected using Model-based Analysis of ChIP-Seq 2 (MACS2, http://liulab.dfci.harvard.edu/MACS/ (14 May 2017, date last accessed)) with default parameters. Images of NGS peaks were generated using Integrative Genomics Veiwer (IGV) (http://software.broadinstitute.org/software/igv/ (14 May 2017, date last accessed)). The accession number of the NGS data is DRA005236. […]

Pipeline specifications

Software tools ELAND, MACS, IGV
Application ChIP-seq analysis
Organisms Gallus gallus