GSM2335501: CIVIT_seq_total_1; Saccharomyces cerevisiae; OTHER
For reverse transcription(RT), primer oBZ181 and RNA were denatured and annealed in (50 mM Tris-Cl pH 8.6, 60 mM NaCl, 10 mM DTT). dNTPs (4mM each final) and MgCl2 (20 mM final), RNAsin Plus (Promega; N2611), and AMV RT (Promega; M5108) were added, and RT was carried out at 42 °C for 1h. RT product was ethanol precipitated and resuspended in water, then treated with RNase T1 and RNase A in (50mM Tris pH7.5, 150mM NaCl) for 1h at 37°C. Full length RT products were purified on an 8% TBE-urea PAGE gel. cDNA was circularized with circLigase (Epicenter) and PCR amplified using oRP1 and barcoded reverse primers. Pooled barcoded libraries were sequenced with 40nt paired-end reads on an Illumina NextSeq.
GEO Accession
GSM2335501