Qubit 1x dsdna high sensitivity assay

Single cells isolated from digested bones were stained with ReadyProbes cell viability imaging kit, blue/green (Thermo Fisher Scientific, catalog # R37609) and manually counted using a hemocytometer under an EVOS M7000 microscope (Thermo Fisher Scientific). Immediately following cell counting, samples were processed using Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index) as described in the manufacturer’s instructions (10X Genomics). In brief, aiming for 8000 cells per library, single-cell suspensions with more than 70% live cells were loaded onto Chromium Controller (10X Genomics) to generate gel beads-in-emulsions. Then, copartitioned cells were lysed, primers were released from gel beads, and barcoded full-length cDNA was produced and amplified. 3′ gene expression libraries were generated from cDNA by fragmentation, end repair, A-tailing, adaptor ligation, and index PCR amplification. The concentration and size distribution of final libraries were assessed by Qubit 1X dsDNA high sensitivity assay (Thermo Fisher Scientific, catalog # Q33231) and the fragment analyzer system (Agilent). Libraries were sequenced either on a NextSeq500 or NovaSeq 6000 (Illumina) with paired-end mode (read1: 28 cycles, read 2: 90 cycles, i7: 10 cycles, i5: 10 cycles) to generate a minimum of 20,000 read pairs per cell.

Viral enrichment of clinical extracts, in vitro culture isolates, and the synthetic SARS-CoV-2 positive control spiked into negative culture supernatant was performed using the Illumina RNA Prep and Enrichment with the Respiratory Viral Oligo Panel (RVOP) v2 (Illumina, United States). This probe-based capture technique was selected as it was designed to generate near full length SARS-CoV-2 genomic sequences with even coverage in mutagenic regions. RNA denaturation, first and second strand cDNA synthesis, cDNA tagmentation, library clean-up and normalisation were performed according to the manufacturer’s instructions. Individual libraries were pooled in 3-plex reactions for probe hybridisation based on each samples SARS-CoV-2 viral load. The final probe hybridisation step was held overnight at 58°C. The enriched library was purified, and the concentration and fragment size were quantified using the Qubit™ 1x dsDNA High Sensitivity Assay (Thermo Fisher Scientific, United States), and Agilent High Sensitivity D1000 ScreenTape assay on the Agilent 4200 Tapestation (Agilent, Germany), respectively. The libraries were sequenced using 2 × 74 bp runs on the Illumina MiniSeq™ or iSeq (Illumina, United States) and multiplexed with the aim of producing 2 × 10⁶ raw reads per library.