title: GSM8703725: Multiplexed Lungs and Hilar Lymph Nodes 1, T-cells, TCR; Homo sapiens; OTHER
library_construction_protocol: Explanted lungs and HLN from study participants undergoing re-transplantation were obtained after receiving approval from our Institutional Review Board. Explanted lungs from research-consented organ donors whose lungs were rejected for clinical transplantation were obtained following approval from our institutional Committee for Oversight of Research and Clinical Training Involving Decedents. Following transplant, lung and HLN tissues were cross sectioned, placed into a tissue cartridge in zinc formalin fixation solution for 48 to 72 hours. Sections were then placed in 70% ethanol prior to paraffin embedding. Samples were embedded within 1 week of transplant. The tissue was then manually cut into fine pieces and transferred to a GentleMACS Dissociator (Miltenyi Biotec) for 30 minutes at 37°C. Following dissociation, tissue was filtered in the following order: first through a stainless-steel mesh filter, then through a 100-micron filter, then through a 70-micron filter. The suspension was then centrifuged at 1500 rpm for 5 minutes. Cells were then counted and frozen back at -80°C for future analyses. Sorted cells were captured for single-cell RNA and T cell receptor (TCR) sequencing using the 10x Genomics 5' assay per manufacturer instructions. Libraries were sequenced using the Illumina platform.