title: Illumina NovaSeq 6000 sequencing; Single cell RNA sequencing of CellTagging lung organoids
design: Single cell RNA sequencing of CellTagging lung organoids
library_construction_protocol: All tubes and pipette tips were pre-washed in 1X HBSS with 1% BSA to prevent cell adhesion to the plastic. 3D cultures (spheroids, LPOs, iBTOs) were removed from Matrigel using a P1000 pipette tip and vigorously pipetted in a 1.5 mL Eppendorf tube to remove as much Matrigel as possible. Tissue was centrifuged at 300g for 3 minutes at 4ºC, then excess media and Matrigel was removed. Tissue was digested to single cells using 0.5 mL TrypLE (Invitrogen, Cat#12605010) and incubated at 37ºC for 30 minutes, adding mechanical digestion with pipette every 10 minutes. After 30 minutes, trypsinization was quenched with 1X HBSS + 1% BSA. Cells were passed through a 40 µm filter (Bel-Art Flowmi, Cat#136800040) and centrifuged at 300g for 3 minutes at 4ºC. Cells were resuspended in 1 mL 1X HBSS + 1% BSA and counted using a hemocytometer, centrifuged at 300g for 3 minutes at 4ºC, and resuspended to a final concentration of 1,100 cells/µL. If samples were planned for combined submission, cells would be cryopreserved in CryoStor CS10 (Biolife Solutions, Cat#210102). If cryopreservation was used, cold CryoStor CS10 solution was added to the cells and mixed thoroughly and transferred to a cryovial. The cells were incubated at 2-8ºC for 10 minutes, then cryopreserved in using an isopropanol freezing container for 24 hours, then transferred to liquid nitrogen. Cells were thawed in a 37ºC water bath, washed in DMEM/F12 (Corning, Cat#10-092-CV) with 10% FBS, centrifuged at 300g for 5 minutes,and supernatant was removed. The pellet was then washed in HBSS + 1% BSA, then resuspended in 1 mL fresh HBSS + 1% BSA, passed through a 40 µm filter (Bel-Art Flowmi, Cat#136800040), and counted using a hemocytometer. Cells were centrifuged at 300g for 3 minutes at 4ºC and resuspended to a final concentration of 1,100 cells/µL. Approximately 100,000 fresh or thawed cells were put on ice and single cell libraries were immediately prepared at the 10X Chromium at the University of Michigan Sequencing Core with a target of 10,000 cells per sample. BTO Line Establishment and Culture: BTO lines were cultured as previously described. Briefly, distal lung tissue was dissected into 1 cm3 chunks and washed with sterile PBS. Tissue was digested using 1 mL Dispase (Corning, Cat#354235) for 30 min on ice. Digestion was then quenched with 1 mL 100% FBS (Thermo Fisher, Cat#35-015-CV) for 15 min on ice following by washing in 1 mL media containing DMEM-F12, 1% Penicillin-streptomycin (Thermo Fisher, Cat#15140122) and 10% FBS for 10 min on ice. Tissue was then mechanically dissociated further using a scalpel, forceps, and vigorous pipetting with a P1000 to remove mesenchyme from bud tip epithelium. Dissociated tissue was transferred to a 15 mL conical tube, washed with DMEM-F12 media described above and spun at 300g for 3 min at 4°C. After each wash, supernatant and separated mesenchyme was removed and fresh DMEM-F12 was added. These steps were repeated 3-5 times until all visible mesenchyme is removed. On final wash, remaining epithelium was transferred to a 1.5 mL microcentrifuge tube and cells were resuspended in Matrigel (Corning, Cat#354234). Cells were plated into 15 µL droplets and allowed to solidify. BTOs were fed 3F media (36) every 3 days and were passaged using 27G needle every 10-14 days. Airway Differentiation: Paradigm Airway differentiation was carried out as previously described. Briefly, BTOs were exposed to dual-SMAD activation (DSA) via 100ng/mL BMP4 (R&D Systems, Cat#314-BP-050) and 100ng/mL TGFβ1 (R&D Systems, Cat#240-B-002) in 3F media for 3 days. On the fourth day, BTOs were exposed to dual-SMAD inactivation (DSI) via 1µM A8301 (APExBIO, Cat#3133), 100ng/mL NOGGIN (R&D Systems, Cat#6057), 10µM Y-27632 (APExBIO, Cat#B1293) and 500ng/mL FGF10 (lab purified – see below) in serum-free basal media for 18 days (media changed every 3 – 4 days) with needle-passaging as needed. CellTag V1 lentivirus was thawed on ice and combined 1:1 with DSI media. 21-day differentiated BTOs were collected for transduction by dislodging Matrigel droplet with P1000 and transferring organoids into a 1.5 mL microcentrifuge tube. Samples were needle passaged by 2 passes through a 27G needle and spun down at 300g for 3 min at 4°C. Cells were resuspended in 1 mL of DSI media + 6µg/mL of polybrene and incubated at 37°C for 15 min with agitation every 2 minutes. Cells were again spun down at 300g for 3 min at 4°C and resuspended in 300 µL virus + DSI. Cells were incubated at 37C for 6hr with agitation every hour. Following transduction, cells were spun down again at 300g for 3 min, resuspended in Matrigel at 1:4 dilution, and fed with 0.5 mL DSI media. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5,000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5,000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used.