GSM7774128: 221122L18_exp221120_MS95_SCGEuri-00nM-BE_1hr-5uM-EU-05min-chase_polyA-EU-enrich-RNA-seq; Schizosaccharomyces pombe; Saccharomyces cerevisiae; RNA-Seq
S. cerevisiae and S. pombe cells were separately pelleted and snap-frozen in liquid nitrogen. Pellets were thawed in ice-cold PBS and mixed. 30 µl of cells in PBS were added to 300 µl TRI Reagent (Zymo Research) and lysed by bead beating using a Fastprep 24. Cell debris was pelleted (14k rpm, 1 minute) and the supernatant recovered. RNA was then extracted using the direct-zol RNA microprep kit (Zymo Research). mRNA was enriched using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490). EU-mRNA was then enriched using the Click-iT Nascent RNA Capture Kit (invitrogen) with the following modifications to the kit protocol. 5 µl Dynabeads MyOne Streptavidin T1 (invitrogen) per sample were washed 1x 50 µl wash buffer 2 (kit component J) and then resuspended in the 50 µl wash buffer 2 + 5% (v/v) Denhardt's reagent and incubated at room temperature (10 minutes). Blocked beads were then washed 3x in 50 µl wash buffer 2 and resuspended in 5 µl wash buffer 2. 12.5 µl Click-iT RNA binding buffer (kit component G) and 0.2 µl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) was then added to 12.3 µl mRNA, mixed, and incubated at 70 °C (5 minutes). 5 µl of blocked and washed beads were then added to each 25 µl mRNA reaction, mixed, and incubated (room temperature, 30 minutes). Beads were then washed 4x with 100 µl wash buffer 1 (kit component I), 2x 100 µl SDS wash buffer (1% SDS, 5 mM TRIS-HCL, 1 mM EDTA), 4x with 100 µl wash buffer 2 and resuspended in 5 µl wash buffer 2. Indexed sequencing libraries were generated using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, #E7775). Biotin-EU-mRNA bound to 5 µl streptavidin beads was immediately used as the input for cDNA synthesis on the beads. cDNA synthesis reactions were pipetted every 10 minutes on the thermocycler to keep Streptavidin beads in suspension. Beads were removed prior to the cDNA clean-up step. Libraries were pooled and then sequenced by paired-end (2x150bp) Illumina sequencing. spike-in normalised polyA EU-enriched RNA-seq