Saccharomyces arboricola Ribo-seq RPFs repB
Lysates containing 30g RNA, as measured using a Qubit 4.0 fluorometer, were diluted to 200L in polysome lysis buffer. Lysates were digested in the presence of 15U RNase1 for 45 min at room temperature. RNA was subsequently purified from lysates before undergoing PNK end repair. Ribosome protected fragments were size selected on 15% urea PAGE gels. Enriched fragments were reverse transcribed into cDNA containing illumina compatible adapter sequences before contaminating rRNA sequences were depleted using EIRNABios custom designed biotinylated depletion oligos for yeast samples. Samples were subsequently amplified by PCR for the generation of Illumina compatible cDNA libraries. Ribosome-protected fragment (RPF) reads had the following structure:5' - QQQ - RPF sequence - NNNNNNNN - AGATCGGAAGAGCACACGTCTGAA - 3'. Where, QQQ (3 nt): untemplated nucleotides introduced during library preparation, RPF sequence: ribosome-protected fragment, NNNNNNNN (8 nt): Unique Molecular Identifier (UMI), AGATCGGAAGAGCACACGTCTGAA: 3 Illumina adapter sequence. For proper reads preparation do: 1. Removal of 3nt untemplated 5' nucleotides and 3' adapter (cutadapt -u 3 -a AGATCGGAAGAGCACACGTCTGAA) and then 2. Removal of 3' 8nt UMI (cutadapt -u -8).
SUB16034207
S. arboricola Ribo-seq repB