title: Illumina HiSeq 4000 paired end sequencing; Post-transcriptional buffering of low nascent RNA transcription by Puf5
design: Post-transcriptional buffering of low nascent RNA transcription by Puf5
library_construction_protocol: Single yeast colonies were picked and inoculated overnight in 5 mL of YPD media at 30°C. The next day, cultures were diluted in 20 mL of YPD media to an OD595  of ~0.15 and grown at 30°C until an OD595 of 0.6-0.8. From each cell culture, 10 mL were collected for RNA extraction (no treatment) and 1 mL of cell culture was diluted in 4 mL of YPD media containing 0.25 μg/mL of Dox and incubated overnight at 30°C. The following day, the overnight cultures were diluted in 40 mL of YPD media containing 0.25 μg/mL of Dox to an OD595 of 0.2 and incubated at 30°C to an OD595 of 0.6-0.8. From each cell culture, 10 mL were then collected for RNA extraction (t0) and the remaining cell culture was diluted in 40 mL of YPD media containing 40 μg/mL of Dox and 1mM Aux to an OD595 of ~0.2. The cell cultures were then further incubated at 30°C and additional samples were taken at 90 and 240 min (t90, t240).  Cells were grown in 100 ml of YPD (30°C and 200 rpm) to mid-log phase (OD600nm 0.5-0.6). Cultures were then split and either DMSO or 3 µg/ml of thiolutin (Abcam ab143556, reconstituted in DMSO) were added. Cells were sampled at time-points 0, 15, 30, and 60 minutes. Total RNA was extracted using the hot phenol method (Schmitt ME, Brown TA, Trumpower BL. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990;18: 3091–3092) 50 ng per sample was reversed transcribed using a polyT30 oligo containing a 8bp barcode for later identification and a unique molecular identifier (UMI). RT used the template switching. After the RT step, all reverse transcribed samples were pooled into a single 2 mL Eppendorf tube and DNA was purified with Ampure XP (supplier, ordering info) beads at 1:1 vol/vol. Purified DNA was eluted in 17 μL H2O and treated with exonuclease (New England Biolabs, ordering info missing). Subsequently, cDNA was amplified and the final PCR product was purified with Ampure XP beads at a volume to volume ratio of 0.8. Finally, 0.8ng cDNA was tagmented in five replicates. Quality of library was assessed on an Agilent Tapestation.