Single-nucleus RNA sequencing of positional airway organoids derived from human fetal lung at 4 anatomic regions (trachea, bronchus, NCA, distal)
Single-nucleus RNA sequencing of positional airway organoids derived from human fetal lung at 4 anatomic regions (trachea, bronchus, NCA, distal)
Human lung tissue research was reviewed and approved by The University of Michigan Institutional Review Board (IRB). Normal, de-identified human fetal lung tissue was obtained from the University of Washington Laboratory of Developmental Biology. Tissue was shipped overnight in UW-Belzer's solution (Thermo Fisher, Cat#NC0952695) on ice and was processed for experiments or fixation within 24h. Normal, de-identified, donor human adult lungs not eligible for transplant were obtained from Gift of Life Michigan Donor Care Center. For organoids, proximal trachea, bronchus, non-cartilaginous airway, or distal tissue was dissected from tissue using a scalpel and forceps and subsequently minced for 2 min into small chunks (<1mm3). Tissue was washed with 1X PBS, spun down at 300g for 3 min at 4°C, resuspended in Matrigel, replated in 20 µL droplets and allowed to solidify. Cultures were fed with 0.5 mL FANY media for 1 week, changing media every 3-4 days, after which they were passaged using TrypLE (Invitrogen, Cat#12605010), for 30 min with mechanical dissociation every 10 min. Dissociation was quenched using DMEM/F-12 (Corning, Cat#10-092-CV) after which cells were spun down at 300g for 3 min at 4°C, resuspended in Matrigel and fed with 0.5 mL FANY media. Cultures were collected for single-nucleus sequencing by pipetting organoids from Matrigel droplet after P2, transferring to a micro-centrifuge tube and gently pipetting to remove excess Matrigel. Cultures were spun down at 300g for 3 min at 4°C and excess supernatant was removed before flash-freezing sample with liquid nitrogen. Preparation was performed as described previously in accordance with 10X Genomics' protocols (CG000124 Rev E for organoids). Briefly, snap-frozen tissue was minced into smaller fragments using a scalpel and then added to lysis buffer (Thermo Fisher Scientific, Cat#PI28324). Tissue was homogenized using a pellet pestle 15 times then incubated for 1 minute in lysis buffer. The cell suspension was filtered through a 33 μm strainer. Suspension was centrifuged at 500g for 5 mins at 4°C. Supernatant was removed and the pellet was washed with PBS + 1% BSA, centrifuged at 500g for 5 mins at 4°C; pellet was washed and centrifuged again. An added debris removal column step was used for the adult tissue.  Permeabilization for ATAC-seq samples was performed by incubating the pellet in 0.1X lysis buffer and incubated for 2 minutes. The suspension was then centrifuged at 500g for 5 mins at 4°C; supernatant was removed and pellet was resuspended in diluted nuclei buffer. Immediately after tissue dissociation, single nucleus libraries were prepared on the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns PN-1000283 was used with a target of 7500 cells.