Miseq paired end platform

For ITS2 amplicon-based DNA sequencing, 0.25 grams of each sample was aseptically weighed and subjected to total DNA extraction in a biosafety cabinet using DNeasy PowerSoil Kit (Qiagen, Germany) and then resuspended utilizing sterile water (UltraPure Distillated Water, Invitrogen, Carlsbad, California, USA). Contamination between samples was excluded using Ultrapure DNA free water as a separate blank control.
As requested by the sequencing service, samples and controls were checked for ITS2 DNA amplicon sequencing eligibility using specific tailed ITS3 and ITS4 primers for the ITS2 region. The DNA concentration required for metagenomics (range: 3–10 ng/μl) was determined by a Qubit spectrophotometer (Invitrogen, Carlsbad, California, USA).
ITS2 barcode sequencing and Sanger sequencing were performed using the NGS sequencing service offered by the BMR Genomics Company, Padua, Italy. Libraries were prepared using Nextera XT DNA Library with sequencing performed via the Illumina MiSeq Paired-End platform.
Sequences were uploaded on GenBank with the BioProject ID: PRJNA851016.

CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺ T cells were prepared as described above. The T cells were lysed with Isogen-LS (NIPPON GENE) and stored at − 80 °C. The lysates were sent to Repertoire Genesis Inc. (Ibaraki, Japan) for next-generation sequencing, which was performed as previously described^{51 (link)}. Briefly, total RNA was converted to complementary DNA (cDNA) with the SuperScript reverse transcriptase (Invitrogen). Double-stranded cDNA was synthesized and ligated with a 5′ adaptor oligonucleotide, then cut with the SphI restriction enzyme. Next, the double stranded cDNA was amplified through polymerase chain reaction using primers specific for the adaptor and TCRα constant region. The sequencing was performed with the Illumina MiSeq paired-end platform (2 × 300 bp). Data processing was performed with the repertoire analysis software developed by Repertoire Genesis Inc^{52 (link)}. TCR sequences were assigned with a dataset of reference sequences from the international ImMunoGeneTics information system database (http://www.imgt.org). The percentage of sequence reads with TRAV, TRAJ, TRBV, and TRBJ genes was calculated. The Circos plots were produced using the Circos software package^{53 (link)}. The Shannon–Weaver index shows the diversity and is defined as follows. $$\mathrm{Shannon}-\mathrm{Weaver}\mathrm{index}=-{\sum }_{\mathrm{i}-1}^{\mathrm{S}}\left(\frac{{\mathrm{n}}_{\mathrm{i}}}{\mathrm{N}},\ln ,\frac{{\mathrm{n}}_{\mathrm{i}}}{\mathrm{N}}\right)$$