GSM6008508: CTR RS Rep2; Saccharomyces cerevisiae; OTHER
The formaldehyde fixation reaction was quenched by adding a buffer containing glycine (20 mM HEPES-KOH pH 7.4 at 25°C, 100 mM KCl and 2 mM MgCl2, 250 mM glycine) to the cell pellet to neutralise any remaining formaldehyde. The reaction was performed on ice following rigorous cell pellet re-suspension. Upon quenching completion, the cells were pelleted by a centrifugation at 4°C, 5,000×g for 5 minutes. Supernatant was discarded and the cell pellet re-suspended in the same buffer but without added glycine, to reduce glycine contamination. Cells were pelleted once more as described above, supernatant discarded and the cell pellet re-suspended in a small volume of the same buffer without added glycine. Cell suspension was flash-frozen by dripping in liquid nitrogen and stored at -80°C. Frozen cell pellets were further disrupted using a cryo-mixing mill set at 27Hz for 2 minutes under liquid nitrogen temperature. The resultant powdered cell grindate was further re-suspended in a buffer containing 20 mM HEPES-KOH pH 7.4 at 25°C, 100 mM KCl, 2 mM MgCl2, 5 mM DTT, 1× EDTA-free protease inhibitor and 1 U/μl of RNaseOUT RNase inhibitor. The suspension was clarified from cell debris by centrifugation at 4°C, 13,000×g for 5 minutes. The resultant clarified lysate was used for further translation complex isolation by separation via sedimentation over a discontinuous sucrose gradient (10-20% w/v, 50% w/v), allowing particles co-sedimenting with ribosomes or faster (Translated Pool, TP; also total translated, tt) to collect in the bottom 50% w/v sucrose cushion. The tt pool was subsequently subjected to a limited RNase I digestion followed by another round of sedimentation through a continuous 10-40% w/v sucrose gradient. Fractions containing ribosomal small subunit (SSU), mono(ribo)somes (RS) and di(ribo)somes (DS) were identified using in-line UV absorbance readout upon sedimentation completion and isolated. RNA in the isolated fractions has been de-crosslinked using hot acid phenol extraction and ethanol precipitation, followed by the construction of TCP-seq libraries. Approximately 1 μg of the SSU, RS and DS de-crosslinled RNA was used to construct TCP-seq libraries generally according to the published methods (Archer, et al., Nature 2016, 535: 570-574; Shirokikh, et al., Nature Protocols, 2017, 12:697-731; Janapala et al., JoVE, 2021, e62639). Briefly, RNA was end-repaired with T4 bacteriophage polynucleotide kinase, 3'-polyadenylated with Escherichia coli poly(A) polymerase, primed with a split-adaptor circularisation DNA oligonucleotide and converted into cDNA using SuperScript IV reverse transcriptase. The resultant cDNA was circularised using CircLigase II and the original RNA degraded with A/T RNase mix. The circularised cDNA was then PDD-treated (Archer, et al., BMC Genomics 2014, 15:401; Archer, et al., Current Protocols in Human Genetics 2015, 85:11.15.1-11.15.36) using a set of probes designed based on the previously-encountered ribosomal(r)RNA TCP-seq library contaminants. Upon the PDD treatment, the cDNA was amplified for 14-28 cycles with primers containing indices and structures compatible with Illumina's TrueSeq Small RNA Sample Preparation Kit. The amplified libraries were size-selected using non-denaturing agarose gel electrophoresis separation and cleaned-up with phenol:chloroform extraction and ethanol precipitation. The resultant amplified and indexed libraries were paired-end sequenced to 150 nt read length with Illumina HiSeq 2500.  RNA-seq libraries were made using the same fixed cell material as the TCP-seq counterparts and included Total cell lysate (TCL; also Total, T) and Translated Pool (TP; also total translated, tt) fractions. ~1 μg of the RNA obtained from each T and tt was directed for making rRNA depleted RNA-seq libraries using GENEWIZ services and the following general approach. The rRNA was depleted using an rRNA removal kit. The VAHTS Total RNA-seq (HMR) Library Prep Kit for Illumina was used for library preparation. The rRNA-depleted RNA was fragmented and reverse-transcribed. First cDNA strand was synthesised using ProtoScript II Reverse Transcriptase with random primers and Actinomycin D. The cDNA second-strand was synthesised using Second Strand Synthesis Enzyme Mix including dACG-TP/dUTP. The double-stranded cDNA was bead-purified and then treated with End Prep Enzyme Mix to repair both ends and create dA-tailing in one reaction, followed by a T-A ligation to add amplification adaptors to both ends. Size selection of the adaptor-ligated DNA was then performed using beads, and fragments of ~400 bp (with the approximate insert size of 300 bp) were recovered. The dUTP-labelled second strand was digested with Uracil-Specific Excision Reagent enzyme. Each sample was then amplified by PCR using Illumina's P5 and P7 index-containing primers. The PCR products were cleaned up using beads and assessed by Nano quality control (Nano QC) to identify library concentration. High-throughput sequencing libraries were then multiplexed and sequenced similarly to the TCP-seq libraries, to obtain at least 100 M reads per each multiplexing barcode. TCP-seq with PDD rRNA depletion, RNA-seq with RNA-stage rRNA depletion