ChIP-seq assay for RocS binding to the Streptococcus pneumoniae genome
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ABSTRACT:
ORGANISM(S): Streptococcus pneumoniae
PROVIDER: GSE129717 | GEO | 2019/04/14
REPOSITORIES: GEO
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Project description:Purpose: The goal of this study is to identify genes selectively associated with C. elegans CEBP-1. Methods: We generated transgenic animals expressing FLAG-tagged CEBP-1 in a cebp-1(tm2807) mutant background (cebp-1(0); juIs418 [Pcebp-1::FLAG::CEBP-1::cebp-1 3’UTR]) and then immunoprecipitated FLAG-CEBP-1-associated DNA fragments using anti-FLAG antibodies (M2 anti-FLAG magnetic beads; Sigma). We collected mixed stage worms grown at 20˚C on NGM plates followed by 2% formaldehyde and sonicated the samples as described (Mukhopadhyay et al., 2008). We next generated ChIP-seq DNA libraries. Briefly, both ChIPed DNA and input genomic DNA were ligated to specific adaptors and amplified by barcode primers followed by sequencing on the Illumina HiSeq-2000 platform. We performed two independent ChIP-seq experiments, with parallel genomic DNA controls prepared from the same strain. We conducted peak-calling using CLC genomics workbench 6.0 (CLCbio). To define genes associated with the peaks, we used the annotation of transcription start site (TSS) and transcription end site (TES) from WS220 and annotated the peak if it overlapped the gene or the 3 kb upstream of the TSS. We then manually confirmed the peaks and associated genes using UCSC browser and update to WS252. Results: We found 209 CEBP-1 ChIP-seq peaks in the genome that were associated with 212 coding genes. Through motif discovery tools, we also found that CEBP-1 binds conserved DNA motifs. Conclusions: The conservation of C.elegans CEBP-1 binding motif supports functional parallels between C. elegans CEBP-1 and vertebrate C/EBPs.
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