GSM9099887: T-4235; Homo sapiens; OTHER
RNA was extracted from postmortem human brain tissue obtained from the New York Brain Bank, including the cingulate cortex and substantia nigra of individuals with Parkinson's disease, dementia with Lewy bodies, or without neuropathological findings (controls). Each tissue sample was dissected fresh-frozen at autopsy and immediately processed. RNA integrity was assessed, and only samples with an RNA integrity number (RIN) greater than 7 were used. For in-house α-chain library preparation, 500 ng of total RNA per sample served as input. For RepSeq+ libraries prepared by iRepertoire, 5 µL of RNA was submitted per sample. For α-chain–only libraries, we followed the iRepertoire Bulk Reagent Universal User Manual (V20200818) using HTAIvc barcode kits. After reverse transcription and amplification, libraries were pooled, spiked with 10% PhiX, and sequenced on an Illumina NextSeq 550. For RepSeq+ libraries, iRepertoire used a targeted amplicon approach to amplify TCR α and β chains simultaneously. The protocol employed gene-specific primers for the constant (C) and variable (V) regions, with unique molecular identifiers (UMIs) incorporated during reverse transcription. Following SPRIselect bead purification steps, a second round of amplification used V-gene primers. Final libraries incorporated Illumina dual-index adapters, a 10-nt UMI, and an 8-nt internal barcode for sample tracking. Sequencing captured regions spanning from framework 1 to the constant region, including CDR1, CDR2, and CDR3.