title: GSM9381127: healthy donor DP-sorted single cell TCR-seq; Homo sapiens; OTHER
library_construction_protocol: For single cell RNA and TCR-sequencing, resected surgical specimens were dissected and grossly identified as tumor or as “non-malignant” kidney. Once received, these tissues were immediately digested with DNAse I and Collagenase IV in RPMI media with mechanical dissociation at 37°C with 5% CO2 for 1 hour. Residual erythrocytes were lysed with ACK lysing buffer (Thermofisher) for 5 minutes and quenched with 10-fold excess PBS. Single cell suspensions were then strained through a 100um filter and counted for viability before downstream experiments such as flow cytometry or scRNAseq. Patient and healthy donor blood were collected in parallel. Peripheral blood mononuclear cells (PBMCs) were processed with Ficoll (Cytiva), isolated by gradient centrifugation and cryopreserved to be later thawed for validation experiments. Sample processing for single-cell gene expression (scRNA-seq) and T cell receptor V(D)J clonotypes (scTCR-seq) was done using the Chromium Single Cell 5′ Library and Gel Bead Kit (10x Genomics), following the manufacturer's user guide. CD3+CD4+, CD3+CD8+, and CD3+CD4+CD8+ T cells were sorted from digested tumor and non-malignant tissues, or Ficoll-purified and previously cryopreserved PBMCs, into 500 ul of PSA/0.04% BSA for loading onto 10X. After Gel Bead-in-Emulsion reverse transcription (GEM-RT) reaction and clean-up, a total of 13 cycles of PCR amplification were performed to obtain sufficient cDNAs used for both RNA-seq library generation and TCR V(D)J targeted enrichment followed by V(D)J library generation. TCR V(D)J enrichment was done per manufacturer's user guide using Chromium Single Cell V(D)J Enrichment Kit, Human T cell (10x Genomics). Following library preparation, sequencing was performed on an Illumina NovaSeq 6000 on S4 flow cells. Patient samples were processed and pooled by sorted T cell populations in parallel during library preparation and sequenced on the same flowcell to minimize batch effects.