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Protocol publication

[…] Sequences (fastq format) were first mapped with tophat () using the following criteria: -m 1 -g 1 --microexonsearch --no-closure-search -I 500000 (command line: tophat -m 1 -F 0 -g 1 --microexon-search --no-closure-search -G ../exon20110528mm.gtf --phred64-quals -I 500000 -o ZT4_2_tophat.out /data/analysis/fasta/mm9 /data/sequence/Nascent_ZT4_2.fastq). About 65–70% of the Nascent-Seq sequences uniquely mapped to the mouse genome, even though no rRNA removal has been performed. Uniquely mapped sequences from the tophat output files (bam format) were then used for further analysis. Wig files, used for signal visualization with the Integrated Genome Browser (), were created as described in UCSC website (ftp://hgdownload.cse.ucsc.edu/apache/htdocs-rr/goldenPath/help/bedgraph.html) and normalized to uniquely mapped reads. The normalization factor used for normalization is indicated in the last column of the . [...] Sequences from the different libraries (fastq format) were first mapped to the mouse genome (version mm9) using bowtie () with the command line: bowtie –q –a –-best –m 1. Only those that mapped uniquely to the mouse genome were used for further analysis, and their number has been used for normalization to compare signal difference between libraries.ChIP-seq libraries were analyzed with the MACS algorithm () by comparing the treatment sample (BMAL1 or CLK ChIP) to the control sample (Input) using the following criteria: effective genome size = 1.89 × 109, tag size = 36, band width = 80, model fold = 5, p-value cutoff = 1 × 10−5. Significant peaks were computationally assigned to a gene. Briefly, a peak located between the transcription start site and the transcription start end of a gene was assigned to that gene, regardless of the ChIP-Seq peak position. The other peaks, referred as intergenic, were assigned to the gene with the closest transcription start site. Confirmation of this computational gene assignment was then confirmed by manual inspection for the 211 CLK:BMAL1 peaks. Visualization of the ChIP-seq signal was performed using the wig output file (from the MACS analysis) and the IGB browser.Overlap between CLK and BMAL1 DNA binding peaks has been determined computationally using all significant peaks coordinates. Any overlap between the two peaks (even of one nucleotide) was considered significant. Quantification of the signal has been extracted from the raw data (number of reads per bp) normalized to sequencing depth of each library. For most experiments (e.g., ), signal was binned using a 25 bp window.Quantification of the number of e-boxes within BMAL1 and CLK DNA binding peaks has been performed computationally using peak fasta sequences. Enrichment has been calculated using the number of e-boxes found at a fixed position from the peak center divided by the expected number of e-boxes. The maximal window size (difference from the fixed position and the peak center) was 500 nt, as the number of expected e-boxes dropped to the background at this window size.Motif analysis has been performed using MEME suite (http://meme.sdsc.edu/meme/intro.html), using a 100 bp sequence for each peak (peak center ± 50 bp). Parameters were as follow: -dna -mod anr -nmotifs 20 -minw 6 -maxw 30. The background model contained the same nucleotide distribution as the input file. Significant motifs were then analyzed using TOMTOM from MEME suite. […]

Pipeline specifications

Software tools Bowtie, IGB, MEME Suite, Tomtom
Application ChIP-seq analysis
Organisms Mus musculus