GSM3640004: Msn5_t15; Saccharomyces cerevisiae; RNA-Seq
RNA was purified using a high-throughput RNA isolation protocol (Dye et al. 2005). Briefly, cells were washed in ddw and incubated with Proteinase K (Epicentre MPRK092) and 1% SDS at 70℃ to release the RNA. Cell debris were precipitated by centrifugation in the presence of KOAc precipitation solution. Finally the RNA was purified from the supernatant using nucleic acid binding plates (UNIFILTER plates, catalog #7700-2810) and was stored with RNAse-inhibitor (Murine #M0314L) at -80°C. RNA-libraries were prepared as previously described (Klein-Brill et al. 2019). Briefly, RNA was reverse transcribed using  SmartScribe enzyme (TaKaRa) and in the presence of  oligo-dT RT primers with a 7bp barcode and a 8bp UMI (a gift from Ido Amit). Barcoded samples were then pooled and purified using SPRI beads X1.2 (AMPure XP). DNA-RNA molecules were tagmented using Tn5 transposase (loaded with oligo Tn5MEDS-A) and 0.2% SDS was used to strip off the Tn5 from the DNA (Picelli et al. 2014). followed by a SPRI X2 clean up. NGS sequences were added to the tagmented DNA by PCR (KAPA HiFi HotStart ReadyMix 2X - KAPA 33 Biosystems KM2605, 12 cycles). Finally, DNA was purified using double SPRI (X0.65  SPRI to clean large fragments followed by X0.8 SPRI beads).
GEO Accession
GSM3640004