Illumina NovaSeq 6000 sequencing; scRNA-seq of canalicular stage human fetal lung cultured under air-liquid interface
scRNA-seq of canalicular stage human fetal lung cultured under air-liquid interface
For scRNA-sequencing of Lung ALI explant cultures, the outside rind of n = 3 explants at each timepoint (0, 3, 6, 9 and 12 days) was micro dissected and minced using a No.1 Scalpel, discarding the center of the explant, which appeared necrotic in later timepoints. Pooled explants at each timepoint (0, 3, 6, 9 and 12 days) were dissociated using the Neural Tissue Dissociation Kit (P) (Mitenyi Biotec, Cat#130092628). Briefly minced explant tissue was resuspended in Mix 1 and incubated at 37ºC for 15 minutes. Mix 2 was then added and incubated for 10 minutes at 37ºC. Cells were agitated by P1000 pipetting and then returned to the incubator for additional 10 minute incubations at 37ºC, followed by P1000 pipetting until a single cell suspension was achieved (~30 minutes). Obtained single cell suspension was filtered through a 70 µm Flowmi Cell Stainer (Sigma, Cat#BAH136800070) and then resuspended in Red Blood Cell Lysis Buffer (Roche, 11814389001) for 15 minutes at 4ºC. After Red Blood Cell Lysis, cells were washed twice with 2mL 1X HBSS + 1% BSA and then resuspended in Cryostor-CS10 (Sigma, Cat#C2874) for storage in liquid nitrogen. All Lung ALI explant culture samples were thawed and co-submitted for sequencing on the same day. Thawing of cells prior to sequencing consisted of adding 1:1 increments of RPMI + 10% FBS drop-wise, with 1 minute pauses every time the volume doubled, until a total volume of 32 mLs was achieved. Cells were then pelleted and resuspended in 1 mL HBSS + 1% BSA and passed through a 40 µm Flowmi Cell Strainer (Sigma, Cat#BAH136800040), counted on a hemocytometer and submitted at 1000 cells/µl in HBSS + 1% BSA to the University of Michigan Advanced Genomics Core for library preparation by the Chromium Next GEM Single Cell 3' GEM, Library and Gel Bead Kit v3.1 (10x Genomics, Cat#PN1000128) targeting 7500 cells.  For ALI explant cultures (2725-2, 2725-3, 2725-4, 2725-5) small pieces (< 0.5 cm diameter) of canalicular stage human lung (122 days post conception) were dissected from distal regions and placed on Nucleopore Track-Etched Membrane disks (13 mm, 8 µm pore, poly-carbonate) (Sigma, Cat#WHA110414) floating on top of 500 µl of human lung ALI explant media (Advanced DMEM/F-12 (Thermo Fisher, Cat#12634010), 2 mM Glutamax (Thermo Fisher, Cat#35050061), 15 mM HEPES (Corning, Cat#25060CI), 1X B27 Supplement (Thermo Fisher, Cat#17504044) 1X N-2 Supplement (Thermo Fisher, Cat#17502048), 100U/mL penicillin-streptomycin (Thermo Fisher, Cat#15140122)) in a 24-well tissue culture plate (Thermo Fisher, Cat#12565163). Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 7500 cells. Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (10x Genomics, Cat#PN1000128) following the manufacturer's protocol. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 7500 cells. Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (10x Genomics, Cat#PN1000128) following the manufacturer's protocol.