scRNA-seq  pancreatic tumor
Oligoconjugated antibodies were diluted with BD Stain Buffer (cat.554656) to volume of 110l. The freshly prepared single-cell suspension was centrifuged and resuspened in 110l of BD Stain Buffer. Then, mixed 100l of single cell suspension and antibody solution. After 45 minutes incubation on ice, the labelled samples were washed with 2mL of BD Stain Buffer for subsequent RNA extraction. Single-cell capture was achieved by a random distribution of a single-cell suspension across over 200000 microwells through a limited dilution approach. Beads with oligonucleotide barcodes were added to saturation to ensure that a bead was paired with a cell in a microwell. Cell lysis buffer was used to release polyadenylated RNA molecules and hybridize RNA to the beads. Then, beads were harvested into a single tube for reverse transcription. Upon cDNA synthesis, each cDNA molecule was tagged on the 5 end with a molecular index and a label indicating its cell of origin. Sequencing was performed with Illumina NovaSeq according to the manufacturer's instructions