Low rna input kit

Tumor RNA samples (1 ng input) were reverse transcribed to cDNA. Primer-specific regions were then linearly amplified through a multiplexed target enrichment approach using the Low RNA Input Kit (NanoString). We used both the Human chimeric antigen receptor (CAR)-T Panel (770 genes plus 10 internal reference genes) and the Human Immune Profiling panel (730 genes plus 40 internal reference genes), each containing probes for immune-related and housekeeping genes. Reaction products were hybridized to cartridges on the NanoString MAX/FLEX Prep Station, which were then scanned on the Digital Analyzer. Read counts were normalized to panel-specific reference genes that were selected using geNorm (20 (link)). Each panel was normalized independently and then merged results were prepared for downstream analyses in nSolver 4.0 with the Advanced Analysis plugin (version 2.0.134). To account for overlapping panel probes, nSolver uses a scaling method that takes the ratio of geometric means of all common probes between the two CodeSets. This ratio was then multiplied to the individual probe counts for both common and unique probes in the subsequent CodeSet. Counts for common probes were averaged in the merged data, while unique probe counts were adjusted by the ratio factor. The resulting values were used for downstream analyses.

Organoids were infected with SARS-CoV-2 and dissociated and stained for FACS as described above in a BSL3-certified biosafety cabinet. Cells were fixed with 4% PFA for 1 hr at RT to inactivate virus as described above for decontamination out of the BSL3 facility. Cells were sorted for live, single cell, EPCAM⁺CD45⁻ cells using a BD FACSAria II SORP and RNA was extracted using the protocol described by Russell et. al^{78 (link)}. Following RNA extraction, quality was assessed via Agilent Bioanalyzer and cDNA was synthesized using Hs HostReponse LI Primers (Nanostring, XT-HHR-12), Coronavirus Panel Plus (Nanostring, CORONAPP-12) and Low RNA Input Kit (Nanostring, LOW-RNA-48) per manufacturer’s instructions. Following hybridization, transcripts were quantitated using the nCounter^® SPRINT Profiler. Samples were run by the LCA Genome Core at University of California, San Francisco.