title: Illumina HiSeq 2000 paired end sequencing; Measurement of Isoform Specific Turnover using sequencing (MIST-Seq) after transcriptional arrest in rpb1-1 strain of budding yeast
design: Measurement of Isoform Specific Turnover using sequencing (MIST-Seq) after transcriptional arrest in rpb1-1 strain of budding yeast
library_construction_protocol: Inoculate a 100 ml culture to OD 0.1 from a 5 ml of overnight growth of a single yeast colony in appropriate growth medium. Grow the temperature sensitive strain in permissive temperature (24C) to OD 1.0 and then apply heat shock by addition of hot (65C) growth medium to the growing cells to bring the culture temperature to 37C.  Harvest the cells at the required time points after applying the heat shock. To extract total RNA yeast cells were lysed by vortexing with glass beads in 2ml tube with FastPrep. RNA was then cleaned by phenol:chlorophorm extraction followed by ethanol precipitation and DNase treatment. In summary 10 microgrammes of total RNA from budding yeast and 1 microgramme of total RNA from fission yeast was fragmented, purified, retrotranscribed, end-repaired, ligated, amplified and finally size selected.  Full protocol is describe in "An efficient method for genome-wide polyadenylation site mapping and RNA quantification" Wilkening et al., 2013