title: GSM5097245: Calu3_Enriched_VH; Homo sapiens; RNA-Seq
library_construction_protocol: Infected cells were lysed in TRIzol (Invitrogen) and RNA was extracted using Direct-zol RNA Miniprep kit (Zymo Research) as per manufacturer's instructions. The VSP pool contained ~30 oligonucleotides that span all SARS-CoV2 genes (at least one oligo per gene and nearly 1 oligo per 1kb of the genome). Given our goal to additionally enrich for potential HVC events, we used two approaches, enrichment methods 1 (5'->3' host-virus chimeras) and enrichment method 2 (5'->3' virus-host chimeras). First strand cDNA synthesis reaction: To capture and enrich for viral, virus-host or host-virus transcripts, we first set up a 20 µl reverse transcription (RT) reaction using 100 ng of mRNA isolated from SARS-CoV2 infected using Superscript III reverse transcriptase. We used 2 pmol T7-P5-VSP oligo pool (for enrichment 1) or 50 pmol of T7-P5-Oligo dT (for enrichment 2) as “gene-specific primer” for RT reaction. After combining all the components as per the recommended protocol (Thermo Fisher, Catalog# 18080044 and 18064014), we incubated the entire reaction mixture at 25°C for 15 minutes followed by 50°C for 30 minutes for SSIII. Next, we inactivated the reaction by heating at 70°C for 15 minutes. To remove RNA/complementary RNA to the cDNA, we added 1 µl of PureLink™ RNase A (20 mg/mL) (Invitrogen Catalog# 12091021) and 1 µl (5 units) of RNase H (abmgood catalog# E018) and incubated at 37°C for 1 hour. We then purified cDNA using 1X Mag-Bind TotalPure NGS (Omega Bio-tek, Catalog# M1378-01) as per manufacturer's instructions and eluted the cDNA in 15 µl of sterile water. To further remove the excess single-stranded short oligos, we treated the purified RT reaction with 1 µl of Exonuclease I (NEB, Catalog# M0293L) at 37°C for 30 minutes. Next, we added excess sterile water to sample to get a total volume of 40 µl. The reaction was them purified with 1X Mag-Bind TotalPure NGS beads and eluted in 15 ul of sterile water. Second strand cDNA synthesis and In vitro Transcription: Following this, we performed second strand synthesis using the NEBNext Ultra II Non-Directional RNA second strand synthesis module as per the suggested protocol (NEB, Catalog# E6111L). The synthesized DNA was purified via 1X Mag-Bind TotalPure NGS beads and eluted in ~12 ul of sterile water. 10 ul of this was then used as an input for T7 polymerase mediated In vitro Transcription (IVT) using the NEB HiScribe T7 High Yield RNA Synthesis Kit (NEB, # E2040S). Briefly, all the components were mixed as mentioned in the kit protocol and incubated at 37°C (lid at 50°C) for 16 hours. The reaction was eluted in 20 µl of sterile water after a round of 1X Mag-Bind TotalPure NGS bead cleanup. This newly transcribed RNA was quantified using a Nanodrop and to improve the hybridization kinetics and enhance signal, 500ng of the amplified RNA was fragmented by RNA Fragmentation reagent in a total reaction volume of 10 µl as per specifications (Thermo Fisher, Catalog# AM8740). Final reverse transcription and PCR enrichment of the library: Next, to generate final enriched libraries, we performed reverse transcription of the fragmented RNA with 50 pmol of P7-N6 for enrichment (1) and 2 pmol of the P7-VSP primer pool for enrichment (2), respectively, using SSIII reverse transcriptase as per the steps mentioned above. After RT, the reaction mixture was purified using 1X Mag-Bind TotalPure NGS beads and eluted in 20 µl of sterile water. 5 µl of this RT reaction was saved for running on a Bioanalyzer and to perform a qRT-PCR validation assay. The remaining 15 µl was used to PCR amplify the library by using high-fidelity Q5 DNA Polymerase (NEB, Catalog# M0491L) for 16 cycles using Universal primer and unique indices (NEB Catalog# E7335L, E7500L) in a total of in a 50 µl reaction volume. Finally, the amplified and enriched library was purified using the 0.8X Mag-Bind TotalPure NGS beads, quantified by Bioanalyzer/Tape station and then sequenced using Illumina platform.