PU-seq of S. cerevisiae: NT RNH201-iAID ELG1-AID pol2
Genomic DNA samples were isolated with Quiagens Blood & Cell Culture DNA Midi Kit, following the manufacturers instructions. Then, ribonucleotides incorporated in the DNA were hydrolyzed with an alkali treatment of 0,3M NaOH at 55C for 2 hours. The fragments were separated by electrophoresis in an alkali agarose gel, as described above. The gel was stained with Diamond Nucleic Acid Dye (Promega), and the ssDNA fragments between 100 and 500 bp were then excised from the gel and purified in NucleoSpin Gel and PCR Cleanup (Macherey-Nagel) columns following the manufacturers protocol. The ssDNA was then quantified with Qubit ssDNA assay Kit (Thermo Fisher Scientific). Sequencing libraries were prepared using NextFlex Rapid Directional RNA-seq library prep Kit (Perkin-Elmer) from the strand synthesis step, following the manufacturer's instructions. DNA was treated with the NEBNext End Repair module (New England Biolabs) and then, the NextFlex protocol was resumed from the adenylation step, following the provided instructions. Finally, libraries were quantified with Qubit HS dsDNA kit (Thermo Fisher Scientific), an Agilent Bioanalizer and qPCR with SYBR Premix Ex Taq (Takara). Libraries were sequenced in a NextSeq 500 platform (Illumina).