Genechip fluidics station 450

T_FH cells (CD4⁺PD-1⁺CXCR5⁺) sorted from 8–10-month-old mice were pooled to generate 5 TC and 3 B6 samples. Total RNA was extracted with RNeasy Mini kit (Qiagen). 200 ng of total RNA per sample was used to generate labeled cDNA fragments with GeneChip^TM WT cDNA Labeling Kit (Thermo Fisher) that were hybridized to Affymetrix MTA 1.0 microarrays (Thermo Fisher) with Thermo Fisher reagents. Microarrays were processed with the GeneChip 3000 7G scanner and GeneChip Fluidics Station 450 (Thermo Fisher). Raw CEL files were normalized by the RMA algorithm with Partek Genomic Suite 6.6 (Partek). SYBR green (Biorad)-based qPCR was performed on cDNA synthesized from sorted T cell subsets with primer sequences shown in Supplementary Table 4. Normalization to housekeeping genes Hmbs or Ppia was carried out with the 2^−ΔΔCt method. A custom nCounter Gene Expression CodeSet panel (Supplementary Table 2) was generated by NanoString Technology. Using 50 ng of total RNA per sample, cartridge preparation and scanning was carried out according to manufacturer’s instructions using the NanoString Prep Station and nCounter Digital Analyzer. Data was analyzed with nSolver Analysis software, version 2.5 with normalization utilizing positive and negative control probes as well as housekeeping genes.

Microarray analysis of 16 Cln3^Δex7/8 brain tissue samples treated with vehicle or GalCer (four males and four females in each group) was performed using the GeneChip Mouse Genome 430 2.0 array platform from Affymetrix (Affymetrix Inc., CA, USA) representing over 45,000 probe sets) (Fig 1). Samples were prepared and microarrays processed using the GeneChip™ 3’ IVT PLUS Reagent Kit (Thermo Fisher Scientific, MA, USA) according to manufacturer instructions. Briefly, 100 ng of total RNA were labeled, fragmented, and then hybridized to arrays. After washing and staining using GeneChip Fluidics Station 450 (Thermo Fisher Scientific, MA, USA), arrays were scanned with the GeneChip Scanner 3000 7G (Thermo Fisher Scientific, MA, USA). Cell Intensity Data (CEL) files were generated with the Affymetrix GeneChip™ Command Console (AGCC) software version 3.2 (Thermo Fisher Scientific, MA, USA).

To isolate total RNA, sorted cells were flash frozen in PBS immediately after sorting and stored at −80 °C prior to RNA extraction. QIAzol Lysis Reagent (Qiagen) was added to the cells, and RNA was isolated and purified using the RNeasy kit (Qiagen). The concentration was measured on a NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was examined using the 2200 TapeStation System with Agilent RNA ScreenTapes (Agilent Technologies). Total RNA was amplified using the GeneChip WT Pico Kit (Thermo Fisher Scientific) generating biotinylated sense-strand DNA targets. The labeled samples were hybridized to human Clariom D arrays (Thermo Fisher Scientific). Washing and staining was performed by the GeneChip Fluidics Station 450 and the scanning was performed using the GeneChip Scanner 3000 (both Thermo Fisher Scientific). All cell populations were generated in triplicate. All data analysis was performed in RStudio. Raw data were normalized using the RMA algorithm implemented in the limma R-package. Adjusted p-values were calculated using the Benjamini-Hochberg method. Data were visualized using ggplot2 and pheatmap R-packages.