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Protocol publication

[…] ormalized to that of GAPDH. All gene expression data represents the mean of at least three technical replicates., PCR primers were designed to amplify the genomic cleavage site for a given sgRNA. Resulting PCR products were subjected to Sanger sequencing. Sequencing traces were used for editing quantification using a previously described publically available tool., Human erythroid H3K27ac ChIP-seq was obtained from Xu et al and mouse erythroid H3K27ac ChIP-seq was obtained from Kowalczyk et al and Dogan et al. We uniformly processed all the datasets using the same pipeline with the same criteria to call super-enhancers. Specifically, we started from raw reads and realigned each dataset with Bowtie2 with the default parameters. We then removed duplicate reads with the Picard Suite. To call the peaks we used MACS2 in the narrow mode. Finally to call the super-enhancers we used the ROSE algorithm with the default parameters. Using these settings, peaks closer than 12.5 kb are stitched together and then ranked based on the H3K27ac intensity. To assign super-enhancers to genes we used again ROSE with default settings. In particular, the tool reports three categories of genes for each super-enhancer: 1) overlapping genes - genes for which the gene body region overlaps a super-enhancer; 2) proximal genes - genes close to a SE considering a window of 50kb; 3) closest gene - closest gene considering its TSS and the center of the super-enhancer. To generate a Venn diagram of genes for super-enhancer dat […]

Pipeline specifications

Software tools Bowtie2, Picard, MACS
Organisms Mus musculus, Homo sapiens