title: Single-cell sequencing samples for chemically defined, serum-free, and xeno-free differentiation of alveolar type 2 cells
design: Single-cell sequencing samples for chemically defined, serum-free, and xeno-free differentiation of alveolar type 2 cells
library_construction_protocol: For dissociation, spheroids were collected and centrifuged at 200g for 4 minutes and resuspended in a dissociation mix of  0.125 mg/mL Collagenase (Merck, C9891), 1 U/mL Dispase II (Thermo Fisher Scientific, #17105041) and 0.1 U/μL DNase (Merck, D4527) mixed in DMEM/F12 (Dulbecco's Modified Eagle's Media: Nutrient Mixture F-12; Thermo Fisher Scientific, #11330-057) for 20 minutes at 37°C on a DLAB MX-T6-Pro Roller at 10 RPM. Dissociation mix was neutralised using 1:3 dilution with basal media and centrifuged at 200 RCF for 5 minutes. Spheroids were resuspended in dissociation mix consisting of 0.05% trypsin-EDTA (Gibco, #25300062) and 0.1 U/μL DNase and placed at 37°C for 10-15 minutes, resuspending manually every 3 minutes. Dissociation mix was neutralised using 10% foetal bovine serum solution in DMEM/F12 at 1:3 dilution. Cells were centrifuged at 300 RCF for 5 minutes, and then counted. Nucleic acid extraction was obtained following Evercode WT v2.3 Parse Biosciences protocol. RT transcription was performed within each cell following single cell combinatorial barcoding. Following barcoding, cells were lysed and nucleic acid molecules extracted.  Single-cell RNA-seq libraries were generated using the Evercode WT v2.3 kit (Parse Biosciences). 8 sublibraries were generated.