4sU-tagging, ribosome profiling and ATAC-Seq

https://www.virologie.uni-wuerzburg.de/virologie/ags-virologie/ag-doelken/research/

1. Principle of 4sU-tagging: 4-thiouridine (4sU) is added to cell culture medium to start metabolic labelling of newly transcribed (nascent) RNA. 5 to 60 min later total RNA is isolated. Following thiol-specific biotinylation, total RNA is separated into labelled nascent and unlabelled pre-existing RNA. All three RNA fractions are suitable for down-stream analyses including qRT-PCR, microarrays and RNA-seq.

2. Principle of ribosome profiling: Following a mild cell lysis, a stringent RNAse digest is performed. The short fragment of RNA molecules actively being translated at the time of cell lysis is protected from RNAse treatment within the ribosome and can then be recovered, cloned and sequenced. Thereby, a detailed picture of translational activity at the time of cell lysis is obtained

3. Chromatin accessibility profiling by ATAC-Seq

https://www.nature.com/articles/s41596-022-00692-9 https://www.illumina.com/techniques/multiomics/epigenetics/atac-seq-chromatin-accessibility.html

• The assay for transposase-accessible chromatin with sequencing (ATAC-Seq) is a popular method for determining chromatin accessibility across the genome. By sequencing regions of open chromatin, ATAC-Seq can help you uncover how chromatin packaging and other factors affect gene expression. ATAC-Seq does not require prior knowledge of regulatory elements, making it a powerful epigenetic discovery tool. It has been used to better understand chromatin accessibility, transcription factor binding, and gene regulation in complex diseases, embryonic development, T-cell activation, and cancer. ATAC-Seq can be performed on bulk cell populations or on single cells at high resolution.

• How Does ATAC-Seq Work? In ATAC-Seq, genomic DNA is exposed to Tn5, a highly active transposase. Tn5 simultaneously fragments DNA, preferentially inserts into open chromatin sites, and adds sequencing primers (a process known as tagmentation). The sequenced DNA identifies the open chromatin and data analysis can provide insight into gene regulation. Additionally, ATAC-Seq can be combined with other methods, such as RNA sequencing, for a multiomic approach to studying gene expression. Subsequent experiments often include ChIP-Seq, Methyl-Seq, or Hi-C-Seq to further characterize forms of epigenetic regulation.