Ncounter sprint profiler machine

Total RNA from sorted T cell populations (n=4-12 mice/group) was extracted with mini RNA-easy Kit (Qiagen). Equal amount of total RNA from different cells was used for the assay. Hybridization reaction was established by following the instruction of manufacture. Aliquots of Reporter Codeset and Capture probeset were thawed at room temperature. Then a master of mix was created by adding 70ul of hybridization buffer to the tube containing the reporter codeset. 8 μl of this master mix was added to each of tubes for different samples, 5 μl (50 ng) of total RNA sample was added into each tube. Then 2 μl of well mixed Capture probeset was added to each tube and placed in the preheated 65°C thermal cycler. All the sample mixes were incubated for 16 hours at 65°C for completion of hybridization. The samples were then loaded into the sample hole in the cartridge and loaded into the NanoString nCounter SPRINT Profiler machine (NanoString). When corresponding RLF running is finished, the raw data was downloaded and analyzed with Nanostring software nSolver 3.0 (Nanostring). mRNA counts were processed to account for hybridization efficiency, background noise, and sample content, and were normalized using the geometric mean of housekeeping genes. Fold changes were calculated comparing the experimental group to their appropriate controls.

Total RNA was extracted as described above. RNA concentrations were quantified and evaluated for purity using a nanodrop spectrophotometer (Thermofisher, # ND-ONE-W). For immune profiling we utilized Nanostring’s 770-gene Mouse nCounter® PanCancer Immune Profiling Panel. Reporter codesets were thawed at room temperature and subsequently mixed with 70 μL of hybridization buffer to form a master mix. From this master mix, 8 μL was aliquoted into PCR tubes. RNA samples were normalized to 10 ng/uL and 5 μL from each sample was added into the PCR tubes. Next, 2uL of Capture probeset was added to each tube. The reaction mixture was then gently mixed, spun down, and loaded into a prewarmed 65°C thermocycler with a heated lid set to 70°C. Samples were allowed to hybridize for 16–20 hours, after which they were immediately placed on ice. At this time, a SPRINT cartridge was thawed and allowed to equilibrate to RT. Samples were then individually loaded into the cartridge and inserted into the NanoString nCounter SPRINT Profiler machine (NanoString nCounter Analysis System, RRID:SCR_021712). Raw data was analyzed using NanoString nSolver 4.0. mRNA counts were processed to account for hybridization efficiency, background noise, and sample content, and were normalized using the geometric mean of housekeeping genes.