title: GSM8608324: HyPR-HN 02_1, TCR; Homo sapiens; OTHER
library_construction_protocol: Bulk RNAseq: Total RNA was extracted from tumor tissue, PBMCs, or cell lines using the Qiagen RNeasy Mini Kit according to the manufacturer's protocol. RNA concentration and quality were assessed using NanoDrop.  Whole exome sequencing: Genomic DNA was extracted from tumor tissue, PBMCs, or cell lines using the Qiagen DNeasy Blood & Tissue Kit following the manufacturer's instructions. DNA concentration and purity were assessed with NanoDrop. Extracted DNA was used for exome library preparation. Single-cell sequecing: Single-cell suspensions were prepared from tumor or PBMCs, and viable cells were loaded into the 10x Genomics Chromium platform for partitioning. RNA from individual cells was reverse-transcribed, and libraries were prepared according to the 10x Genomics protocol. Spatial: Slides were processed through the 10x Genomics Gene Expression workflow with pre-sequencing H&E imaging completed on the Keyence Microscope per 10x Genomics workflows. Next, transcripts were de-crosslinked, hybridized to a probe panel, ligation completed, and probes released for capture on the gridded, barcoded surface of the Visium slide (CytAssist, 10x Genomics, RRID:SCR_024570). After barcoding, the samples underwent library preparation, amplification, and clean-up with quality assessment ensuring that fragments were ~240bp. Bulk RNAseq: RNA was extracted and used for mRNA library preparation with poly(A) enrichment, following Novogene's standard protocol. Library construction and sequencing were performed on the NovaSeq X Plus Series platform with paired-end 150 bp (PE150) reads. Whole exome sequencing: Genomic DNA was extracted and used for library preparation with the Agilent V6 kit, according to Novogene's standard workflow. Library quality was assessed, and sequencing was conducted on the NovaSeq X Plus Series platform with paired-end 150 bp (PE150) reads. Single-cell sequencing: Library preparation was completed at the MCW Mellowes Center (RRID:SCR_022926) with 10x Genomics Chromium Next GEM Single Cell 5' Reagent Kits (10X Genomics, sample dual indexes with cellular and molecular barcodes with VDJ amplification). Target capture of 10,000 cells per sample was used. Prior to sequencing, libraries were quantified and pooled by qPCR (Kapa Library Quantification Kit, Kapa Biosystems). Spatial: Libraries were sequenced at the Mellowes Center Facility on the Illumina NovaSeq6000 with paired end reads per the 10x Genomics protocol and generating 25 million read pairs per spot covered by tissue. The quality control of raw reads was processed by FastQC.