title: GSM4475018: Yeast_rRNA_PolysomeF1.rep2; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: For the samples 1-8 and 17-18-21-22:Saccharomyces cerevisiae BY4741 cells (strains: snR3Δ, snR34Δ snR36Δ, and WT) were harvested via centrifugation at 3000 RPM for 1 minute, followed by two washes with water. RNA was purified from pelleted cells using a MasterPure Yeast RNA extraction kit (Lucigen, MPY03100), according to manufacturer's instructions., For the rest (Fractions): Yeast pellets from 100 mL cultures were washed with 6 mL of ice-cold Polysome Extraction Buffer (PEB), which contained 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 100 U/mL RNAse inhibitors (RNaseOUT, Invitrogen, #18080051). Cells were centrifuged for 5 min at 3,000 g at 4ºC. Washing was repeated by adding 6 mL of ice-cold PEB, followed by centrifugation. Cells were then resuspended in 700 uL of ice-cold PEB, and transferred into pre-chilled 2 mL Eppendorf tubes containing 450 uL of pre-chilled RNAse-free 425-600 μm diameter glass beads (Sigma G8772). Cells were lysed by vortexing for 5 min at 4 ºC, followed by centrifugation at maximum speed at bench centrifuge for 5 min at 4ºC. 10% of the supernatant was aliquoted into Trizol for total RNA isolation, and kept at -80ºC. The remaining volume, corresponding approximately to 8 x 10e8 cells, was subsequently loaded onto the sucrose gradient. Linear sucrose gradients of 10-50% were prepared using the Gradient Station (BioComp). Briefly, SW41 centrifugation tubes (Beckman, Ultra-ClearTM) were filled with Gradient Solution 1 (GS1), which consisted of 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 10% w/v RNAse-free sucrose. The tube was then filled with 6.3 mL of Gradient Solution 2 (GS2) layered at the bottom of the tube, which consisted of 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 50% w/v RNAse-free sucrose. The linear gradient was formed using the tilted methodology, with the Gradient Station Maker (Biocomp). Once the gradients were formed, 400 µL of each lysate was carefully loaded on top of the gradients, and tubes were balanced in pairs, placed into pre-chilled SW41 buckets and centrifuged for 150 min, at 35,000 RPM. Gradients were then immediately fractionated using the Gradient Station, and 20 x 500 uL fractions were collected in 1.5 mL Eppendorf tubes, while absorbance was monitored at 260 nm continuously. Fractions were combined in the following way: the free rRNA (F1), the unassembled subunits (F2), the lowly-translating monosomes (F3) and the highly-translating polysomes (F4). The pooled fractions were then concentrated using Amicon-Ultra 100K columns (Millipore), and washed two times with PEB. The final volume was brought down to 200 µL, and RNA was extracted using TRIzol reagent. Yeast total RNA was first treated with T4 Polynucleotide Kinase (PNK) (NEB M0201S)  in order to remove possible phosphorylated 3'ends before polyA tailing. Briefly, we mixed 1 ul PNK enzyme with rRNAs in a 50 ul reaction with T4 PNK Buffer (10X) and incubated at 37°C for 30 minutes. The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. Dephosphorylated rRNAs were then polyadenylated using E.coli Poly(A) Polymerase, following the commercial protocol. Four different direct RNA libraries were barcoded according to the recent protocol that our lab has recently published [58]. Custom RT adaptors (IDT) were annealed using following conditions: custom Oligo A and B (Table S4) were mixed in annealing buffer (0.01M Tris-Cl pH7.5, 0.05M NaCl) to the final concentration of 1.4 uM each in a total volume of 75 ul. The mixture was incubated at 94 °C for 5 minutes and slowly cooled down (-0.1 °C/s) to room temperature. RNA library for direct RNA Sequencing (SQK-RNA002) was prepared following the ONT Direct RNA Sequencing protocol version DRS_9080_v2_revI_14Aug2019 with half reaction for each library until the RNA Adapter (RMX) ligation step. Per reaction (half), 250 ng total of polyA tailed yeast rRNAs were ligated to pre-annealed custom RT adaptors (IDT) [58] (Table S4) using concentrated T4 DNA Ligase (NEB-M0202T), and was reverse transcribed using Maxima H Minus RT (Thermo Scientific, EP0752), without heat inactivation step. The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. 50 ng of reverse transcribed RNA from each reaction was pooled and RMX adapter, composed of sequencing adapters with motor protein, was ligated onto the RNA:DNA hybrid and the mix was purified using 1X Agencourt RNAClean XP beads, washing with Wash Buffer (WSB) twice. The sample was then eluted in Elution Buffer (EB) and mixed with RNA Running Buffer  (RRB) prior to loading onto a primed R9.4.1 flow cell, and ran on a MinION sequencer with MinKNOW acquisition software version v.3.5.5.. Nanopore Direct RNA Sequencing