title: GSM5825306: wtRat1_1: BY4741 + Rat1-∆NLS-3xFlag_rep1; Saccharomyces cerevisiae BY4741; OTHER
library_construction_protocol: Total RNA was isolated by the standard phenol: chloroform method, and DNA was removed by DNase I treatment. The amount of RNA was quantified in a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and RNA integrity was checked by agarose gel electrophoresis. HT-5Pseq libraries were prepared as previously (PMID: 33870233). In brief, 6 μg of DNA-free total RNA were directly ligated with RNA/RNA oligo containing UMI (RNA rP5_RND oligo). Ligated RNA was reverse transcribed and primed with Illumina PE2 compatible oligos containing random hexamers and oligo-dT. RNA in RNA/DNA hybrid was depleted by sodium hydroxide with 20 min at 65 degree incubation. Ribosomal RNAs were depleted using DSN (Duplex-specific nuclease) with the mixture of ribosomal DNA probes. Samples were amplified by PCR and sequence in an Illumina NextSeq 500 instrument using 60 sequencing cycles for read1 and 15 cycles for read 2. For 5'Cap-seq, 5'capped mRNAs were captured as previous described (cite Pelechano et.al 2016). Specifically, 10 µg total RNA were treated with calf intestinal alkaline phosphatase (NEB, CIP) to remove 5′P molecules (fragmented and non-capped). After purification, mRNA 5'cap were removed by Cap-Clip (Biozyme), resulting in 5'P molecules from previously capped molecules. The following steps is the same as the one described for HT-5Pseq (see above) with variations only for skipping removing ribosomal RNA.