title: GSM7163166: BCR_SR_26; Homo sapiens; OTHER
library_construction_protocol: 25-30 μm thick FFPE tissue sections were obtained from each patient, deparaffinized with xylene, and total RNA and DNA were extracted simultaneously with the AllPrep DNA/RNA FFPE Kit (Qiagen, 80234) following the vendor's instructions. Final DNA and RNA concentrations were measured with Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851) and RNA HS Assay Kits (ThermoFisher, Q32852), respectively. RNA isolated from PaxGene storted frozen blood sample of VILI_5 with PAXgene Blood RNA Kit according to vendor's protocol. Total RNA isolation from T-cells performed with Allprep DNA/RNA Mini Kit according to vendor's protocol. cDNA was synthesized from RNA by using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, 11754250) and Ion Torrent NGS Reverse Transcription Kit (ThermoFisher, A45003) for TCR and BCR sequencing, respectively. T-cell and B-cell RNA was quantified by qPCR with TaqMan Gene Expression Assay, CD247 (20X, Hs00167901_m1) and CD19 (20X, Hs01047413_g1). Final input was normalized to 1 ng of T- and B-cell derived RNA per sample. For samples with low T- and B-cell counts, maximum RNA input was loaded for library preparation. Next-Generation Sequencing (NGS) libraries were prepared using the Oncomine TCR Beta-SR RNA Assay (ThermoFisher, A39359) and the Oncomine BCR IGH SR RNA Assay (ThermoFisher, A45484) according to manufacturer's instructions. Amplified and barcode ligated libraries were purified with AMPure XP Reagent (Beckman Coulter, A63880) and quantified with the Ion Universal Library Quantitation Kit (ThermoFisher, A26217). The library pool was prepared by combining equal volumes of libraries at 50 pmol/L concentration and loaded into Ion 550™ Chip (ThermoFisher, A34537).