Advanced analysis 2

Thawed cell pellets were resuspended in RLT lysis buffer with β-mercaptoethanol at 3,500 cells/μL, and overnight hybridization reactions with the nCounter Immunology Panel (Human V2) Reporter CodeSet and Capture ProbeSet were ran according to the manufacturer’s instructions. Samples were run on the nCounter SPRINT Profiler (NanoString), and mRNA counts were normalized in groups by day and cell type (D0, S1R1D14 mocks, S1R1D14 CD19 CAR) using nSolver 4.0 software (NanoString) and the Advanced Analysis 2.0 software (NanoString), which selects the housekeeping genes that minimize the pairwise variation statistic. Each group has 6 samples, 3 biological replicates for antibody-based isolation and 3 biological replicates for aptamer-based isolation. Using Excel (Microsoft), mRNA probes that gave normalized counts less than 25 for more than 50% of the samples in a group (i.e. 4 or more samples) were removed from the analysis due to being mostly below background. The unadjusted P-values of the LOG₂ fold changes in the probe counts of aptamer-isolated cells over antibody-isolated cells were determined using a paired two-tailed t-test in Excel, and the threshold for significance was calculated using the Benjamini-Yekutieli multiple-testing correction in R software.

The retinal tissue mRNA expressions were profiled and analyzed with the Nanostring nCounter^® Mouse Neuroinflammation panel, which contains 757 neuroinflammation-related mouse genes and 13 internal reference controls (#115000237; Nanostring Technologies, Seattle, WA). Briefly, the total RNA from a single retina was extracted using the RNeasy kit (Qiagen, Hilden, Germany). A total of 100 ng of RNA in a volume of 5 μl was hybridized to the capture and reporter probe set for 16 hr at 65°C according to the manufacturer’s instructions. The individual hybridization reactions were washed and eluted per the protocol at the Biomedical Center at Takara Bio (Kusatsu, Japan), and the data were collected using an nCounter Digital Analyzer (Nanostring). The generated data were evaluated using an internal quality control process, and the resulting data were normalized to the geometric mean of the housekeeping genes using the nSolver 4.0 and Advanced Analysis 2.0 software (Nanostring).
We analyzed retinas from female WT mice (C57BL/6J), rd10 mice, and rd10; Ccl2^−/− mice at P31, each comprising three to four biological repeats. DEGs were defined as genes demonstrating a significant difference in expression at the level of p < 0.05 (adjusted p-value by t-test). Unsupervised hierarchical clustering and a heat map analysis were performed using the nSolver software.