GSM2976182: Rap1-AID degron induction 2 hours 5' seq replicate 2; Saccharomyces cerevisiae; RNA-Seq
Cell pellets were washed once with sterile water and snap-frozen in liquid nitrogen. Total RNA was extracted using the hot acid phenol protocol (acid phenol:chloroform:isoamyl alcohol 125:24:1) and TE-SDS buffer, then precipitated in ethanol with 0.3 M sodium acetate before re-suspension in DEPC-treated sterile water. To obtain libraries representing the 5' ends of polyadenylated and capped transcripts, approximately 5 µg of poly(A)+-RNA together with in-vitro spike ins were first subjected to zinc-mediated fragmentation (Ambion) at 70 °C. The reaction was cleaned up using Qiagen min-elute columns to isolate RNA fragments with mode length of ~200 nt. These fragments were then incubated with shrimp alkaline phosphatase at 37 °C to remove the 5' phosphate groups of non-capped fragments followed by acid-phenol/chloroform extraction and ethanol precipitation. With the exception of a non-decapping control sample, dephosphorylated fragments were next treated with Cap-Clip™ acid pyrophosphatase (CellScript) to remove the 5'-terminal caps from fragments representing the bona fide 5' ends of transcripts. After another round of acid-phenol/chloroform extraction and ethanol precipitation, all samples were treated with T4 RNA ligase 1, high concentration (NEB) to introduce a custom adapter sequence to the 5' un-capped ends of fragments. Excess adapters were removed via a column clean up step. First strand cDNA synthesis was performed using Superscript IV (ThermoFisher) and second strand synthesis was performed using a KAPA HiFi HotStart ReadyMix (KAPA Biosystems). Double-stranded cDNA was quantified by Qubit (Life Technologies) and used as input for library preparation using a KAPA Hyper Prep kit (KAPA Biosystems) and KAPA Single-indexed adapters (KAPA Biosystems). Libraries quantified by Qubit and were sequenced on the Illumina HiSeq 4000 platform with 75-base single-end reads.
GEO
GSM2976182