Glycogen

EV-enriched samples were treated with 4 μg/mL of RNase A (Sigma-Aldrich, Burlington, MA, USA) for 1 h at 37 °C, to eliminate any non-vesicular RNA. TRI Reagent (MRC, Cincinnati, OH, USA) was added to a final volume of 1 mL and incubated at RT for 20 min. Then, 200 μL of a Chloroform and Isoamyl Alcohol dilution (24:1) (Panreac Química SLU, Barcelona, Spain) were added, followed by vigorous shaking and centrifugation at 12,000× g for 15 min at 4 °C. Upper fraction was collected, and RNA was precipitated by adding 2.5 μL of glycogen (Merck) and 500 μL 2-propanol (Merck), followed by incubation at RT for 10 min and further centrifugation at 12,000× g for 10 min at 4 °C. RNA pellet was then washed with 75% ethanol, dried at 95 °C for 3 min and resuspended in 12 μL of nuclease-free water.
The DNA-free DNA Removal Kit (Thermo Fisher Scientific) was used to eliminate any DNA remaining in the samples. Following the manufacturer’s protocol, 0.75 μL of DNase buffer and 1 μL enzyme were added to 7.5 μL RNA sample and incubated at 37 °C for 30 min. A volume of 0.75 μL of DNase inactivation reagent was then added to the reaction, incubated for 2 min at RT and centrifuged for 1.5 min at 10,000× g and RT. The supernatant containing EV-RNA was then transferred to a fresh tube and stored at −80 °C until further use.

Total RNA was isolated from 200 μL of sample using the miRNeasy micro kit (Qiagen) as per the manufacturer’s instructions with one modification: following lysis with Qiazol, glycogen (10 μg, Sigma Aldrich, G1767) was added as a carrier to each sample. Complementary DNA libraries were prepared using the QIAseq miRNA Library Kit with QIAseq miRNA NGS 48 Index IL (Qiagen) as per the manufacturer’s protocol (HB-2157-007 March 2020), with the following modifications: 5 μL of RNA was used as the template and the library amplification increased to 24 cycles. Libraries were analysed using the High Sensitivity DNA chip (Agilent) on the Agilent Bioanalyser 2100 to ensure correct insert size and minimal adapter or primer carryover. Libraries were sent to the Australian Genome Research Facility (AGRF) for 100 bp single end sequencing on the NovaSeq 6000 (Illumina).

Exosomes: RNA from salivary exosomes was isolated using the method described by Prendergast et al. RNA was extracted by adding 750 μL TRIzol LS reagent (Thermo Fisher Scientific) and 200 μL chloroform to 40 μL exosomal sample [37 (link)]. We used 5 μL glycogen (5 mg/mL, Sigma Aldrich, St. Louis, MO, USA) instead of 3 μL, as described in the original protocol. The RNA pellet was resuspended in 32 μL of nuclease-free water. The final concentration of the purified RNA was determined using Qubit Fluorometer 4.
Tissues: TRIzol reagent was used to extract total RNA from OSCC tissue (as per the manufacturer’s protocol). The purity and yield of extracted RNA were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer 4 (Thermo Fisher Scientific, Waltham, MA, USA).

Total RNA was extracted using TRIzol reagent per manufacturer’s instructions (Invitrogen), with the modification of 2 μl of glycogen (20 mg/ml, Sigma) added to the isopropanol precipitation step, which was allowed to proceed overnight at -30°C. The following day, samples were centrifuged for 30 min at 10,000 rpm, 4°C, before completion of the standard procedure. Purified RNA was quantified using the Qubit fluorometer and Quant-IT RNA assay kit per manufacturer’s instructions (Invitrogen). RNA quality was checked using Bioanalyzer RNA 6000 Nano chips per manufacturer’s instructions (Agilent).

RNA was extracted from 1.05 mL of plasma using TRIzol LS (Life Technologies, USA), following the manufacturer’s protocol. During the RNA extraction protocol, glycogen (180 μg; Sigma-Aldrich, USA) was added to each sample to facilitate visualisation of precipitated RNA, and an exogenous miRNA control, syn-cel-miR-39-3p (0.25 fmol; Qiagen, NL), was spiked-in to each sample. RNA was re-suspended in 30 μL of RNase-free water and used immediately or frozen at −80 °C.

Chromatin IP followed by exonuclease treatment was performed using the protocol described by Rhee and Pugh with the following specifics [83 (link)]. Rpb3-FLAG WT and rtr1Δ and Nrd1-TAP WT and rtr1Δ cells were grown to an OD₆₀₀ = 0.8–1 prior to crosslinking with formaldehyde. Immunoprecipitation was performed with 50μL of anti-FLAG agarose or anti-TAP sepharose beads (Sigma). The volume of beads used for immunoprecipitation was optimized by affinity purification followed by mass spectrometry [72 (link), 108 (link)]. Subsequent sample processing steps including exonuclease treatment and sequencing library preparation were performed as previously described [83 (link)].
Micrococcal nuclease (MNase) digest and sequencing was performed through adaptation of the protocol by Wal and Pugh [109 (link)]. Following optimization of the digestion conditions, 15U of MNase was added to a chromatin slurry and incubated with shaking at 37°C for 20 minutes. The digestion was quenched by addition of 50 mM EDTA and 0.2% SDS. The digested DNA was cleaned up through phenol/chorloform extraction followed by ethanol precipitation with 20ug of glycogen (Sigma) as a carrier.
ChIP-exo and MNase library construction, EZBead preparation, and Next-Gen sequencing were completed using standard methods based on the Life Technologies SOLiD5500xl system as previously described [57 (link)].

Glycogen (Type III: from rabbit liver) was purchased from Sigma-Aldrich. 7AW was purchased from Aldrich.