Rna screentape analysis kit

Small RNA isolated from the infected U4.4 cells was quantified and quality-controlled with the 4150 TapeStation System using the RNA ScreenTape Analysis kit (Agilent Technologies, Santa Clara, CA, USA). Triplicates for each timepoint (24–72 h) were pooled and submitted to SciLifeLab (Stockholm, Sweden) for library preparation using the QIAseq miRNA low input kit (QIAGEN, Hilden, Germany), and libraries were sequenced on one flow cell of the NextSeq 2000 with a 101nt(Read1)-8nt(Index1) setup using the ‘P2′ flow cell, which generated 10–20 million reads per sample with a read length of 1 × 100 base-pairs (bps).

Small RNAs were isolated while using the miRNeasy Mini Kit (QIAGEN, Toronto, ON, Canada) following the Quick-Start protocol of miRNeasy Mini Kit manufacturer’s instructions. Briefly, tracheal rings from TOC samples (stored in RNAlater) were collected, lysed in 700 µL of QIAzol reagent (QIAGEN, Toronto, ON, Canada), and homogenized for two minutes using 0.5 mm glass beads (Biospec Products Inc., Bartlesville, OK, USA) and a tissue homogenizer (MP FastPrep-24 Classic Instrument, MP Biomedicals, Solon, OH, USA). The isolated EVs from EV samples were likewise lysed in 700 µL of QIAzol reagent (QIAGEN, Toronto, ON, Canada). The samples were then deproteinized in chloroform and centrifuged for 15 min. at 12,000× g at 4 °C. The upper aqueous portion of the samples was precipitated in 95% ethanol. The samples were sequentially washed and centrifuged with RWT and RPE buffers using RNeasy Mini columns in 2 mL collection tubes (QIAGEN, Toronto, ON, Canada). Finally, the purified RNA was eluted in 27 μL RNase-free water and quality control of RNA was performed using the RNA ScreenTape Analysis kit (Agilent Technologies, Santa Clara, CA, USA) and the Agilent 4200 TapeStation Analysis Software A.02.01 SR1 (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer’s instruction. The samples were then stored at −80 °C.

FFPE tissues were sectioned 3-µm thick for hematoxylin and eosin staining (Dako coverstainer, Agilent, Santa Clara, CA, USA) to ensure appropriate tumor-cell content. We used consecutive sections to extract both genomic DNA and RNA with Recoverall total nucleic acid isolation kit according to the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). We then quantified the purified DNA and RNA with respectively Qubit dsDNA BR assay kit (ThermoFisher Scientific, Waltham, MA, USA) and RNA Screentape Analysis kit (Agilent, Santa Clara, CA, USA).