Protocols

Elongation factor-specific mNET capture

Lea developed ELCAP (Elongation factor-specific mNET capture), an affinity purification method that enables capture of factor-specific RNAPII elongation complexes for single nucleotide resolution occupancy profiling and proteomics. As a proof-of-principle this was used to uncover the genome-wide binding profiles of the two RNAPII associated factors SCAF4 and SCAF8 using ELCAP-seq and the RNAPII complex composition in combination with proteomics.

For a detailed step-by-step protocol see: Gregersen et al., 2021 Cell Reports Methods

TT_chem-seq and DRB/TT_chem-seq

Lea developed TT_chem-seq (transient transcriptome profiling using chemical RNA fragmentation) during her postdoc to profile nascent transcription in cells at high resolution. The technique is based on a short pulse labelling of RNA with 4-thiouridine in combination with high-throughput sequencing.

In combination with the early elongation inhibitor DRB (5,6-dichlorobenzimidazole), TT_chem-seq can be used to measure RNAPII elongation rates in cell culture.

For a detailed step-by-step protocol see: Gregersen et al., 2020 Nature Protocols

A quick method to asses global nascent RNA levels is a dot blot of biotinylated 4sU labelled RNA. We routinely use this method to test 4sU labelling of new cell lines and to track global changes in transcription.

For a detailed protocol see: