![]() Open the Galaxy Upload Manager ( galaxy-upload on the top-right of the tool panel)īy default, Galaxy uses the URL as the name, so rename the files with a more useful name. Import the files from Zenodo or from the shared data library Select the option Create New from the menu.Click on the galaxy-gear icon ( History options) on the top of the history panel. ![]() hands_on Hands-on: Data uploadĬreate a new history for this tutorial tip Tip: Creating a new historyĬlick the new-history icon at the top of the history panel Then, to increase the number of reads that will map to the reference genome (here human genome version 38, GRCh38/hg38), we need to preprocess the reads. We first need to download the sequenced reads (FASTQs) as well as other annotation files. Your results may be slightly different from the ones presented in this tutorialĭue to differing versions of tools, reference data, exte rnal databases, orīecause of stochastic processes in the algorithms. Create heatmap of coverage at TSS with deepTools.Controls for the ATAC-Seq procedure are not commonly performed, as discussed here, but could be ATAC-Seq of purified DNA. If you do not have any control data you can import and edit this workflow, removing all steps with the controls. When you use your own data we suggest you to use this workflow which includes the same steps but is compatible with replicates. For that reason, we will download binding sites of CTCF identified by ChIP in the same cell line from ENCODE (ENCSR000AKB, dataset ENCFF933NTR). Good ATAC-Seq data would have accessible regions both within and outside of TSS, for example, at some CTCF binding sites. CTCF is known to bind to thousands of sites in the genome and thus it can be used as a positive control for assessing if the ATAC-Seq experiment is good quality. Furthermore, we want to compare the predicted open chromatin regions to the known binding sites of CTCF, a DNA-binding protein implicated in 3D structure: CTCF. We also added about 200,000 reads pairs that will map to chromosome 22 to have a good profile on this chromosome, similar to what you might get with a typical ATAC-Seq sample (2 x 20 million reads in original FASTQ). The original dataset had 2 x 200 million reads and would be too big to process in a training session, so we downsampled the original dataset to 200,000 randomly selected reads. The data is from a human cell line of purified CD4+ T cells, called GM12878. ![]() 2013, the first paper on the ATAC-Seq method. In this tutorial we will use data from the study of Buenrostro et al. Paired-end reads are recommended for ATAC-Seq for the reasons described here. The read library is then prepared for sequencing, including PCR amplification with full Nextera adapters and purification steps. During ATAC-Seq, the modified Tn5 inserts DNA sequences corresponding to truncated Nextera adapters into open regions of the genome and concurrently, the DNA is sheared by the transposase activity. A transposase can bind to a transposable element, which is a DNA sequence that can change its position (jump) within a genome (read the two links to get a deeper insight). With ATAC-Seq, to find accessible (open) chromatin regions, the genome is treated with a hyperactive derivative of the Tn5 transposase. ATAC-Seq has become popular for identifying accessible regions of the genome as it’s easier, faster and requires less cells than alte rnative techniques, such as FAIRE-Seq and DNase-Seq. In contrast, a silencer decreases or inhibits the gene’s expression. When transcription factors bind an enhancer and contact a promoter region, the transcription of the gene is increased. An enhancer is a DNA region that can be located up to 1 Mb downstream or upstream of the promoter. It contains binding sites for transcription factors that will recruit the RNA polymerase. A promoter is the DNA region close to the transcription start site (TSS). The method can help identify promoter regions and potential enhancers and silencers. Assay for Transposase- Accessible Chromatin using sequencing ( ATAC-Seq) is a method to investigate the accessibility of chromatin and thus a method to determine regulatory mechanisms of gene expression. Consequently, these factors are also important for the activation and inactivation of genes. Many factors, such as the chromatin structure, the position of the nucleosomes, and histone modifications, play an important role in the organization and accessibility of the DNA. When the DNA is being actively transcribed into RNA, the DNA will be opened and loosened from the nucleosome complex. A nucleosome is a complex formed by eight histone proteins that is wrapped with ~147bp of DNA. ![]() In many eukaryotic organisms, such as humans, the genome is tightly packed and organized with the help of nucleosomes (chromatin).
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