Genomic Run On (GRO): determination of the nascent transcriptional rates in a spt16 mutant

We analyzed the effect of SPT16 on the transcription rates, mRNA stabilities and mRNA levels by doing GRO experiments in a thermosensitive spt16 point mutant (G132D) mutant at 35 ºC comparing with permisive temperature (30 ºC) and a wt treated in the same way. Cells (wt or spt16 mutant) were grown in SC-ura at 30 ºC to 0.25 OD600. At that time α factor was added and let to stand for 160 min in the same conditions. Then each sample (wt or mutant) was separated in two aliquots. One of them remain at 30ºC and the other was incubated at 35ºC. Both aliquots in the presence of α factor for 1h more. Finally, samples were processed for GRO experiment as described in Garcia-Marinez et al (2004). Three replicates of the whole experiment were done.