Raw PacBio sequencing of Spike RBD from cells producing VLPs
The Spike RBD PCR was conducted using Roche HiFi PCR kit according to the manufacturer's instructions. The amplicons were purified with XP beads (Beckman Coulter) and made into sequencing libraries according to PacBios Preparing multiplexed amplicon libraries using SMRTbell prep kit 3.0 (102-141-700) protocol. The amplicons from producer cells, VLPs, and infected cells were barcoded (SMRTbell barcoded adapter plate 3.0 (102-009-200)) and pooled for each technical replicate for a total of four pools. Each pool was further prepared for sequencing using the Sequel II binding kit 3.2 (102-333-300), prior to loading one pool per SMRT cell 8M (101-389-001) on a PacBio Sequel IIe platform. Each pool was loaded at 100 pM and sequenced using the >=3Kb Amplicons application. Movie time was set to 30 hr with a 2 hr pre-extension. Circular consensus sequencing (CCS) and demultiplexing were performed on SMRTLink (v. 11.1.0.166339) Productivity rate (P1) was 60% on average, yielding ~3.2 M reads per SMRT cell.