Illumina MiSeq sequencing; Transcription start site mapping of Saccharomyces cerevisiae grown in YPD media using the nAnTi-iCAGE protocol
Transcription start site mapping of Saccharomyces cerevisiae grown in YPD media using the nAnTi-iCAGE protocol
S. cerevisiae was grown in YPD media, and when the cells reached the exponential phase, cells were collected by centrifugation. The cell material was stored at -80 degrees C prior to RNA isolation. We collected three separately grown biological triplicates. Total RNA was extracted from S. cerevisiae using the standard hot-phenol method. The extracted RNA was additionally purified using the Qiagen RNeasy kit (clean-up protocol, according to manufacturer's instructions). Isolated RNA was quantified using NanoDrop1000 and the quality of RNA assessed on the bioanalyzer (Agilent). Total RNA from biological triplicates was pooled in equal amounts prior to library preparation. The nAnT-iCAGE libraries were prepared using the recommended 5 micrograms of total cellular RNA according to the published protocol in Murata et al (Murata et al 2014, Methods Mol Biol 1164: 67-85. Eight technical replicates were prepared using the following barcodes: ACC, CAC, AGT, GCG, ATG, TAC, ACG, GCT. The libraries were sequenced on MiSeq Illumina platform in single-end 50 base-pair mode.