title: Single-cell transcriptomic profiling of patient-derived pancreatic ductal adenocarcinoma primary cell cultures
design: Single-cell transcriptomic profiling of patient-derived pancreatic ductal adenocarcinoma primary cell cultures
library_construction_protocol: To perform scRNA-seq, PDCs were cultured in two batches, 14 samples in the first batch (R1) and 27 samples in the second batch (R2). Following thawing, each PDC was cultured for a maximum of two passages over approximately two weeks. At that point, cells at 60–70% confluency were harvested, and aliquots of 500,000 cells were pelleted, resuspended in 500 µL of freezing medium (FBS + 10% DMSO; Sigma, D8418), counted, and cryopreserved at -80°C using a Corning CoolCell, for subsequent single-cell transcriptomic analysis. Frozen samples were processed using the Evercode™ Cell Fixation v2 kit (Parse Biosciences, ECF2101) and subsequently barcoded and prepared for sequencing according to the manufacturer's workflow provided with the Evercode™ Whole Transcriptome v2 kit (Parse Biosciences, ECW02130). No direct RNA extraction was performed. During library construction, the cells were permeabilized, and the RNA molecules were barcoded in situ with well-specific barcode sequences. The SPLiT-seq protocol applies three rounds of combinatorial indexing to uniquely label individual cells. Briefly, for each PDC, approximately 2,000 fixed cells were used as input. Cells were first distributed into 48 wells for reverse transcription, during which the first molecular tag was incorporated. They were then pooled and redistributed twice into 96-well plates for the sequential ligation of the second and third indexes. After this indexing process, cells were evenly divided into sublibraries and lysed. The uniquely tagged cDNA molecules were isolated using a streptavidin-coated magnetic bead pulldown, followed by PCR amplification. The cDNA was then enzymatically fragmented, ligated to sequencing adapters, and assigned a sublibrary-specific fourth index during the final PCR step.