mRNA half-life calculations
In order to determine single gene mRNA HLs, transcriptional shut-off was
used following the addition of thiolutin up to 5 µg/mL. Samples were
collected at 0, 5, 12, 25, 45, 60 and 90 min after thiolutin addition.
Samples were analyzed by Northern blot using specific32P-probes as described elsewhere (Pelechano &
Pérez-Ortín, 2008).
To determine the global mRNA HL, a dot-blot strategy was followed as
described in (García-Martínez et al., 2016). Known amounts of total RNA
were spotted onto a nylon membrane and hybridized with a32P-terminal labelled oligo d(T)40probe. RNA samples were collected after thiolutin addition at the same
times as described for individual mRNAs. The detailed protocol is
described in (Benet et al., 2017).