Ncounter single cell gene expression assay

Total RNA was extracted from each 250-300 cell sample using the RNAqueous-Micro Total Isolation Kit (ThermoScientific AM-1931). RNA was eluted into 8 µl of Elution Solution, divided into 2 µl and 6 µl aliquots and stored at -80˚C until prepared for NanoString processing. RNA (6ul) was reverse-transcribed into complementary DNA by adding SuperScript VILO Master Mix (2ul, Invitrogen) as described (Tagliafierro et al., 2016 (link)). All samples also underwent target enrichment, and were stored at 4 °C within 24 hrs prior to using in the nCounter Single Cell Gene Expression assay (NanoString Technologies). NanoString assays were carried out by the Preclinical Genomic Pathology Laboratory at the Lineberger Center at the University of North Carolina School of Medicine, Chapel Hill NC.

Arcturus XT (Life Technologies, Carlsbad, CA, USA) was used to perform laser capture microdissection. Cells showing lentiviral vector infection were captured by hand and transferred onto a cap (Capsuret Macro LCM Caps, Life Technologies, catalog number LCM0211) using a near-infrared laser pulse. RNA was extracted from caps using Single-Cell Lysis Kit following the manufacturer’s instruction (Life Technologies, Grand Island, NY, USA). Given the very low amount of RNA collected, we measured mRNA expression using the nCounter Single-Cell Gene Expression assay following the manufacturer’s instruction (Nanostring Technologies, Seattle, WA, USA).