Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates

Thioguanosine Conversion Enables mRNA‐Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC‐seq DUAL)

“…Here, we examined the potential of 6sG‐modified RNA for oxidative substitution chemistry. We assumed that the conditions that we previously elaborated for quantitative 4sU‐to‐C conversions (TUC‐seq), namely the application of osmium tetroxide in ammonium chloride buffer, would result in the formation of 2‐aminoadenosine, which should be decoded as adenine in RNA‐sequencing experiments. Hence, we incubated chemically synthesized 6sG‐containing short RNAs with OsO 4 /NH 4 Cl at pH 8.9 for 2 hours at 40 °C (Figure ).…”

“…In this study, we have developed the selective and quantitative conversion of 6sG‐ into A′‐containing RNA, where A′ constitutes a 6‐hydrazino purine derivative that is cleanly decoded as adenosine in RNA sequencing experiments. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the new OsO 4 /NH 4 Cl/hydrazine conversion chemistry is fully compatible with the conversion of the frequently applied metabolic label 4‐thiouridine (4sU) into C (TUC‐seq), the combination of both modified nucleosides in dual labeling setups enables high accuracy measurements of RNA decay. A pulse‐pulse dual labeling strategy minimizes or eliminates two major caveats of currently employed pulse‐chase settings, namely the persistence of the labeled nucleotide in the cellular nucleotide pool after removal of the labeling medium on one hand and the internal recycling of modified nucleotides on the other hand.…”

“…In the original protocol, the isolated total RNA was thiol‐specifically biotinylated, followed by separation into newly transcribed and pre‐existing, unlabeled RNA . With TUC‐seq, we have recently published the first protocol that eliminates bothersome RNA enrichment steps by quantitatively converting 4‐thiouridine into native cytosine using oxidative OsO 4 ‐based substitution chemistry, allowing for direct detection of newly synthesized transcripts containing U‐to‐C mutations by sequencing . Two related methods, SLAM‐seq and TimeLapse‐seq, have also been reported; however, both approaches modify 4sU to a non‐native pyrimidine analogue, an S‐alkylated 4sU thioiminoester and an N ‐trifluoroethyl‐modified cytosine, respectively.…”

“…These two pyrimidine derivatives give rise to apparent U‐to‐C mutations in RNA‐seq analysis although one has to keep in mind that downstream processing can be complicated or even impaired by the modification. All three 4sU‐based RNA‐seq methods have been validated independently by meaningful applications, including detection of endogenous 4sU in tRNAs, determination of RNA‐synthesis rates on the basis of single genes, as well as transcriptome‐wide, transcript‐specific RNA turnover mediated by post‐transcriptional gene regulation by microRNAs and N 6 ‐methyladenosine, identification of direct transcription‐factor targets, analysis of new RNAs after heat shock, or cellular response to virus infection at the single‐cell level …”

Sister-chromatid-sensitive Hi-C reveals the conformation of replicated human chromosomes

“…3c). Based on a statistical procedure from Erceg et al 37 , we estimated that wrongly assigned trans sister contacts are below 2 % (Extended Data Fig. 3d,e).…”