Rnase free dnase 1

DNase-treated RNA was isolated from spleens as previously described, following the manufacturer’s protocol [39 (link)]. Briefly, macrophages and splenic cells were subjected to RNA extraction using RiboEx reagent (Geneall, South Korea) and RNeasy® Mini kits (Qiagen, Germany). RNase-free DNase 1 (Qiagen, Korea) was used to remove genomic DNA contamination. The purified RNA was collected and stored at − 70 °C until use. Total RNA was measured using a nano spectrophotometer (Optizen, Korea).

Total RNA was isolated from snap-frozen gastrocnemius using the RNeasy tissue mini kit (Qiagen, Milan, Italy) following the manufacturer’s instructions. The purity, integrity, and yield of RNA were analyzed by Agilent Technologies 2001 bioanalyzer using the RNA 6000 LabChip kit. Total RNA (1 µg) was treated with RNase-Free DNase 1 (Qiagen) and reverse transcribed using the High-Capacity cDNA Reverse Transcription System (Applied Biosystems (Thermo Fisher Scientific), Milan, Italy) according to the manufacturer’s instructions. qRT-PCR assays were performed in 96-well optical plates using Mx3000P LightCycler instrument (Stratagene, Milan, Italy). Each cDNA sample was analyzed in duplicates using gene-specific primers spanning intron/exon boundaries for gene expression quantification (see Table 1 for primer sequences) and Fast SYBR Green Master Mix (Applied Biosystems (Thermo Fisher Scientific), Milan, Italy). The following reaction mixture per well was used: 5 µL of Fast Syber Green 2x (Applied Biosystems), 1 µL of primer mix at a final concentration of 250 nM, 2 µL of RNase-free water, and 2 µL of cDNA. Quantitative normalization of cDNA in each sample was performed using either 18S ribosomal RNA or TATA-box binding protein (Tbp) as an internal control. Relative quantification was calculated using the 2^−∆∆CT method.

Frozen whole brains were homogenized in RLT‐lysis buffer (Qiagen) for 30 s at 30 Hz/s using Qiagen TissueLyzer II with 4–5 single‐use silicon beads per sample and total RNA was extracted using the Qiagen RNeasy Micro Kit. On‐column DNase digestion was performed using RNase‐Free DNase1 (Qiagen) following the manufacturer's instructions, to remove any DNA contamination. RNA concentration and quality were determined using Qubit and Agilent 2100 Bioanalyzer, respectively (mean RIN = 7.79), following which RNA‐Seq libraries were built using the KAPA‐stranded mRNA‐Seq kit with 100 ng total RNA as input from each of the 16 individuals. The samples were subsequently sequenced on an Illumina NovaSeq 6000 to get 151 bp, paired‐end reads at the Centre for PanorOmics Sciences, The University of Hong Kong. An average of 33 ± 2.8 million read pairs were obtained from the 16 RNA libraries sequenced (Table S2).

HIV-1 RNA was extracted from blood plasma samples using the RNeasy Lipid Tissue Mini Kit (QIAGEN) with modifications from the manufacturer’s standard protocol^{50 (link)}. Briefly, 100 µl of blood plasma was efficiently lysed in 1000 µl Qiazol Lysis Reagent (Qiagen). DNA was removed by treating the column with RNase-free DNase 1 (Qiagen) prior to RNA elution in 40 µl nuclease-free water. Reverse transcription and amplification of partial pol gene were performed using the One-Step Superscript III RT/Platinum Taq High Fidelity Enzyme Mix (ThermoFisher Scientific^TM) with the pol-specific primer pair JA269 and JA272^{51 (link)}. First-round PCR products were amplified in a nested PCR with DreamTaq Green DNA Polymerase (ThermoFisher Scientific^TM) using pol-specific primers JA271 and JA270. PCR products were sequenced in both directions with the nested PCR primers using the BigDye terminator kit v1.1 (Applied Biosystems) and the sequences were determined on an ABI PRISM 3130×1 Genetic Analyzer (Applied Biosystems).

Foxp3⁺ Tregs were isolated from spleen and lymph nodes of naive and nephritic FIR-tiger and FIR-tiger-PD-L1^−/− mice. Therefore, CD4⁺ T cells were enriched from single cell suspensions by using the CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). Thereafter, CD4⁺ Foxp3⁺ (mRFP⁺) Tregs were purely isolated by FACS. RNA of 1–2 × 10⁶ Tregs was isolated by the RNeasy Mini Kit (Quiagen) and digested with RNase-free DNase I (Quiagen) according to the manufacturer’s instructions. Gene expression analysis was performed using the NanoString assay with the nCounter Immunology Panel profiling 561 immunology-related genes (NanoString Technologies, Hamburg, Germany) according to the manufacturer’s instructions. The results were analyzed with the nSolver Analysis Software 2.5 (NanoString Technologies). Data were calculated in x-fold changes compared to the corresponding control mRNA and displayed as heat map using the web server Heatmapper^{48 (link)}. Clustering was done using the average linkage and the Pearson distance.