Rneasy micro kit 50

Renal CD4⁺ T cell were enriched with CD4⁺ T cell isolation kit (Miltenyi Biotec) in accordance with the manufacturer’s instructions and CD4⁺ T cells were further purified by FACS ARIA sorting. RNA of CD4⁺ T cells (≤ 5 × 10⁴ cells) was extracted by RNeasy micro kit 50 (QIAGEN, Germany). Samples that passed quality control were amplified with the SMARTer kit and then subjected to second generation sequencing via the Illumina platform. The sequenced results were quality controlled in the using the fastqc package. Samples that passed quality control were removed using the Trimmomatic package to remove poor quality sequences and artificially added plug sequences during sequencing. A mean of 39,039,302 paired-end, 150 nt reads were generated. Bases less than Q30 and adapter sequences of the reads were trimmed, and any reads shorter than 36 nt were removed using Trimmomatic version 0.36 [23 (link)]. The trimmed sequencing results which passed quality control reads were then aligned to the mice reference assembly GRCm39 with “Kallisto” analyzed with “DE-seq2” [24 (link)]. Normalization and differential expression analyses were performed with DESeq2 [25 (link)].

RNA was extracted with the RNeasy Micro Kit (50) (Qiagen) according to the manufacturer’s protocol. RNA quantification was performed with a Qubit fluorometric system (Life Technologies).
For the adjuvant cohort, the Illumina TruSight Oncology (TSO, San Diego, California, USA) 500 assay was used to validate a subset of the identified variants at the DNA level in a preselected set of 523 genes. The TSO 500 analysis was performed in the Biomedical Sequencing Facility at CeMM—Research Center for Molecular Medicine of the Austrian Academy of Sciences (Vienna, Austria).
This approach also allowed for the screening of single-nucleotide variants, small insertions and deletions in 151 genes, amplifications in 59 genes, and gene fusions plus splice variants in 55 genes at the RNA level.

NK cells were sorted from the spleen of both Tox^fl/fl and Tox^fl/flCD122^Cre mice. Total RNA from NK cells was extracted using RNeasy Micro Kit (50) (Qiagen 74004) according to manufacturer's protocol. The extracted RNA was evaluated for purity and quality using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The integrity of the extracted RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the gene libraries were constructed according to the manufacturer's instructions using Single Cell Full Length mRNA-Amplification Kit (Vazyme, N712-03, Nanjing, China) and TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD502-02, Nanjing, China). Transcriptome sequencing and subsequent data analysis were performed at OE Biotech Co., Ltd. (Shanghai, China).