Superscript 3

Small-RNA libraries were generated as described in the step-by-step protocol available at http://bartellab.wi.mit.edu/protocols.html, with slight modifications. Briefly, between 5–10 µg of total RNA was mixed with size-selection markers (18 and 32 nt 5′-radiolabeled RNAs), and for the total-sRNA libraries quantitative sequencing standards were added (0.05 fmol per 1 µg total RNA). The samples were first size-selected, before undergoing subtractive hybridization to remove the 2S rRNA (Seitz et al. 2008 (link)). Degenerate adaptors (each containing four random-sequence nucleotides abutting the ligation junction) were then ligated to the 3′ and 5′ ends of the RNA in the presence of 10% Polyethylene Glycol (PEG 8000, NEB) and 0.5 µL of Superasin (Thermo Fisher Scientific) with T4 RNA Ligase 2, truncated K227Q (NEB) and T4 RNA Ligase 1 (NEB), respectively. Reverse transcription was carried out with SuperScript III (NEB), and the cDNA was PCR-amplified using Kapa HiFi HotStart Ready Mix (VWR). Libraries were sequenced on an Illumina HiSeq platform with 50-nt single-end reads. Oligonucleotides used for markers, subtractive hybridization, adaptors, and primers are listed (Supplemental Table S5).

400 µl of nasopharyngeal swab solubilized in UTM was extracted using EZ1 virus mini kit (Qiagen) into 90 µl elution buffer. An aliquot was heat-inactivated at 60°C for 30 min as required by Ministry of Health (Singapore) to prevent SARS-COV-2 infection and 6.5 µl of the heat-inactivated nucleic acid was reverse transcribed using SuperScript III (Life Technologies) (2 µl 5x FS buffer, 0.5 µl dNTPs, 0.5 µl 0.1M DTT, 0.25 µl SuperScript III) using 0.25 µl of Random Primer mix (NEB) using the following protocol: 65°C 20s, 4°C 60s, 55°C 50 min, 95°C 5 min.