GSM5940935: aCD4_dLN_1; Mus musculus; RNA-Seq
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice. Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1mg/mL Liberase TM (Roche). The cells were then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter). Cells were then stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. Cells were also stained with Sample Tag oligonucleotide-conjugated antibodies in BD® Single-Cell Multiplexing Kit (BD Biosciences) as summarized in Supplementary Table 1. CD8+ T cells were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%. T cells from the spleen of Pmel-1 transgenic mice which stained with Sample Tag oligonucleotide-conjugated antibodies as summarized in Supplementary Table 1 were spiked in after cell sorting. Live cells were stained with Calcein AM (Nacalai) and counted using Flow-Count fluorospheres and a Cytoflex flow cytometer. For single-cell RNA/TCRseq, 18,000-30,000 labeled cells were trapped and reverse-transcribed using the BD RhapsodyTM (BD) according to the manufacturer's instructions. Targeted mRNA, TCRseq and Sample Tag libraries were prepared using according to the manufacturer's instructions (VDJ CDR3 and Sample Tag Library Preparation Protocol) with the following modifications; (1) BD RhapsodyTM Immune Response Panel Mm with supplement panel including Cmpk2, Ifi204, Ifi211, Ifit1, Ifit3, Ifit3b, Ifit3b, Isg15, Mx1, Rsad2, and Usp18 were used for target mRNA amplification. (2) we used original set of primer for TCR amplification as summarized in Supplementary Table 2, and performed additional PCR (3rd PCR, 6 cycles) to introduce unique dual index for sequencing. (3) we used ProNex Size-Selection DNA purification System (Promega) for purification of PCR product. Amplified libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems) and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). Targeted RNAseq and Sample Tag libraries were sequenced on the Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 141 bp read 2) (Illumina) to a depth of approximately 20,000-40,000 reads and 5,000-10,000 reads per cell, respectively. TCRseq libraries were sequenced on the Illumina Novaseq 6000 SP flowcell (67 bp read 1 and 459 or 141 bp read 2) (Illumina) to a depth of approximately 5,000-20,000 reads. Bulk TCR sequencing library was prepared according to the previous report (Tsunoda et al., 2021). In brief, to perform reverse transcription and template-switching, mRNA-trapped oligo-dT-immobilized Dynabeads M270-streptavidin (Thermo Fisher Scientific) were suspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 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], and incubated for 60 min at 42ºC and immediately cooled on ice. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed following the purification of PCR product by an Agencort AM Pure XP kit (Beckman Coulter) at a 0.7:1 ratio of beads to sample and eluted with 20 µL of 10 mM Tris-HCl (pH 8.0). To amplify TCR libraries and add adaptor sequences for the next-generation sequencer, the third PCR was performed. The third-PCR products were purified as second PCR. The products were pooled and then purified and subjected to dual size selection using ProNex size-selective purification system (Promega) and eluted with 25 μL of 10 mM Tris-HCl (pH 8.5). Final TCR libraries, whose lengths were about 600 base pairs were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 141 bp read 2) (Illumina) to a depth of approximately 20,000-40,000 reads and 5,000-10,000 reads per cell, respectively. TCRseq libraries were sequenced on the Illumina Novaseq 6000 SP flowcell (67 bp read 1 and 459 or 141 bp read 2) (Illumina) to a depth of approximately 5,000-20,000 reads.(67 bp read 1 and 140 bp read 2) (Illumina). Only read2 contained the sequence regarding the definition of T cell clones. single-cell targeted RNA-seq / TCR-seq Bulk TCR-seq