title: Illumina NovaSeq 6000 sequencing; Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells
design: Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells
library_construction_protocol: Human embryonic and foetal lung tissues were provided from terminations of pregnancy from Cambridge University Hospitals NHS Foundation Trust under permission from NHS Research Ethical Committee (96/085) and the MRC/Wellcome Trust Human Developmental Biology Resource (London and Newcastle, University College London (UCL) site REC reference: 18/LO/0822; Newcastle site REC reference: 18/NE/0290; Project 200454; www.hdbr.org). Sample age ranged from 4 to 23 weeks of gestation (post-conception weeks; pcw). Stages of the samples were determined according to their external physical appearance and measurements. All the samples used for the current study had no known genetic abnormalities. The isolated tip epithelial cells were embedded in Matrigel (Corning, 356231) and cultured in 48-well plates in self-renewal (SN) medium: Advanced DMEM/F12 supplemented with 1x GlutaMax, 1 mM HEPES and Penicillin/Streptomycin, 1X B27 supplement (without Vitamin A), 1X N2 supplement, 1.25 mM n-Acetylcysteine, 50 ng/ml recombinant human EGF (PeproTech, AF-100-15), 100 ng/ml recombinant human Noggin (PeproTech, 120-10C), 100 ng/ml recombinant human FGF10 (PeproTech, 100-26), 100 ng/ml recombinant human FGF7 (PeproTech, 100-19), 3 μM CHIR99021 (Stem Cell Institute, University of Cambridge) and 10 mM SB431542 (Bio-Techne, 1614). The culture medium was replaced every 2 days and the organoids were usually split 1:3 once per week by breaking them into small fragments. Numbers of replicates are indicated in figure legends. 10 ng/μl recombinant human BMP4 (Peprotech, 120-05) and 10 ng/μl recombinant human TGF-β1 (Peprotech, 100-21) were added to the medium instead of adding SB431542 and Noggin to activate dual SMAD signalling. Four biological replicates of the LinPOS and alveolar organoids were harvested individually and enzymatically dissociated into single cells using TrypLE Express Enzyme. The cell suspension was passed through a 30 μm filter, pelleted, and resuspended in appropriate volume with 0.04% BSA/PBS Single cell RNA seq was carried out using according to 10X Chromium Single Cell V(D)J Kits (v1).