GSM6760149: nonEPAP_Drrp6_R3; Saccharomyces cerevisiae; RNA-Seq
RNAs were prepared with hot phenol extraction method. 400 μg RNA was incubated with MTESA biotin-XX linker (Biotium, BT90066) in the biotinylation buffer (10mM Tris pH7.4-7.5, 1mM EDTA, 400ug RNA, 40ug MTSEA biotin-XX linker) in the dark at 24 °C for 30min with 750 rpm shaking. After biotinylation, Chloroform/Isoamyl alcohol (24:1) was applied to remove excess MTSEA biotin-XX liner. After purification, 200 μL of μMACS streptavidin MicroBeads (Miltenyi Biotec, 130-074-101) was added to bind biotin labelled RNAs and isolate them. The RNAs eluted from streptavidin beads were further cleaned up by RNeasy minElute kit (Qiagen, 74204). To efficiently capture <200 nt fragments from the MinElute spin columns, the amount of ethanol added to the RNA and RLT buffer was increased compared to the manufacturer's recommendation. For a 200 μL sample, 700 μL of RLT buffer and 1,050 μL of 100% ethanol was added, mixed well and applied to the minElute spin columns over three rounds. The remaining protocol was as recommended by Qiagen. The RNA was eluted in 20 μL. Where relevant, in vitro polyadenylation was performed by using E. coli polyA polymerase (EPAP) (NEB, M0276L). Ribosome RNAs were depleted by using RiboMinus Transcriptome Isolation Kit (Thermo Fisher Scientific, 10388792). Library of 3' end sequencing was prepared by using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit REV (Lexogen, 16.24) for Illumina and sequenced with single end 75bp reads by NextSeq 500/550 Mid Output Kit v2.5 (150 Cycles) with advanced sequencing science technology platform (STP) at the Francis Crick institute.