GSM8111863: Pno1 + Dim1-E85A, 18S rRNA 3'-RACE, biol rep 6; Saccharomyces cerevisiae BY4741; RNA-Seq
Cells were harvested between OD 1.0 – 2.0. Ribosomes were purified, ribosomal subunits were separated during purification, and 18S and 25S rRNAs were isolated from from 40S and 60S ribosomal subunits, respectively, by phenol-chloroform-isoamyl alcohol extraction. rRNA was treated with DNase I and size selected on a denaturing TBE-Urea-PAGE gel. rRNA was extracted from the gel by freeze-thawing the gel pieces in RNA extraction buffer (300 mM NaOAc, 1 mM EDTA, and 0.25% SDS) and ethanol precipitated. rRNA 3'-ends were dephosphorylated using rSAP and ligated to either the Universal miRNA Cloning Linker or ligated to a pre-adenylated linker containing a UMI sequence using truncated T4 RNA ligase K227Q to protect the 3'-end of the rRNAs from degradation and to identify the true rRNA 3'-end after sequencing. Linkers were added in 2-fold excess of rRNA ends. 5'-ends of the DNA linkers and the rRNA were deadenylated by 5'-deadenylase and excess linkers were degraded by DNA-specific RecJf exonuclease. rRNA was reverse transcribed using a linker-specific primer and Protoscript II RT to generate the first strand of cDNA. After RNase H treatment, the second strand was synthesized using an 18S or 25S rRNA-specific forward primer, a linker-specific reverse primer, and Q5 high fidelity DNA polymerase. The forward and reverse second strand synthesis primers contained partial P5 and P7 Illumina sequencing adapters. The cDNA was purified either on a denaturing TBE-Urea-PAGE gel and extracted by freeze-thawing in DNA extraction buffer (300 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA) or cleaned up with AMPure XP beads. The purified cDNA was amplified via PCR to generate the final libraries and add the complete P5 and P7 Illumina adapter sequences. Library size and quality were assessed on an Agilent 2100 Bioanalyzer. Validated libraries were pooled at equimolar ratios and loaded onto the NextSeq 500 flow cell.