title: GSM6424157: NaraCOVID_TP4_CD4_021_TCR; Homo sapiens; OTHER
library_construction_protocol: PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C CD4+ and CD8+ T cells were magnetically isolated from PBMC. PBMC were thawed by warming frozen cryovials in a 37°C water bath. Cells were stained for 20 minutes at 4°C with 1st antibody mix (Antibody Mix 1 in Supplementary Table S1). After washing with MACS buffer [0.5% BSA (Nacalai), 2 mmol/L EDTA (NIPPON GENE) in PBS (Nacalai)], Cells were stained for 20 minutes at 4°C with 2nd antibody mix; BD IMag anti-PE Magnetic Particles-DM (Clone E31-1459, BD) and anti-APC-Biotin (clone APC003, BioLegend). Then, the cell suspension was diluted with MACS buffer and set on a magnet plate (DynaMag96SideSkirted, Veritas). The positive fraction was collected as CD4+ T cells. The negative fraction was stained with BD IMag Streptavidin Particles Plus-DM (BD) for 20 minutes at 4°C. Then, the cell suspension was diluted with MACS buffer and set on a magnet plate. The positive fraction was collected as CD8+ T cells and B cells. The counts of CD4+ T cells, CD8+ T cells, and B cells were determined using Flow-Count fluorospheres (Beckman) and a Cytoflex flow cytometer (Beckman) and FlowJo software (version 10.8.1; BD Biosciences). Bulk TCR sequencing library was prepared according to the previous report (Aoki et al., 2023).  In brief, mRNA in T-cell lysates were captured by incubating with 10 ?L of Dynabeads M270 streptavidin (Thermo Fisher Scientific) bound to 2.5 pmol of BioEcoP-dT25-adapter primers for 30 minutes at room temperature under gentle rotation. mRNA-trapped beads were suspended in 10 µL of RT mix and incubated for 60 min at 42ºC to perform reverse transcription (RT) and template-switching. The composition of the RT mix was as follows; 1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP (Roche), 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed using KAPA Hifi Hotstart ReadyMix (KAPA Biosystems), followed by the purification of PCR product using an AM Pure XP kit (Beckman Coulter) at a 0.7:1 beads-to-sample ratio. The third PCR was performed to amplify TCR libraries and add adaptor sequences for the next-generation sequencer. The third-PCR products were purified as second PCR. Final TCR libraries, whose lengths were about 600 base pairs, were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems), and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). TCR libraries were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina) using NovaSeq 6000 S4 Reagent Kit v1.5 (200 cycles, Illumina). Only read2 contained the sequence regarding the definition of T-cell clones. Oligonucleotides and reaction conditions for library preparation were summarized in Table S2 and S3, respectively.