GSM6754359: AT1 cell ablation day 10; Mus musculus; RNA-Seq
lungs were inflated with an enzyme dissociation solution (450U/mL Collagenase I (Worthington, LS004197), 5U/mL Dispase (Corning, 354235), and 0.33U/mL DNase I (Roche, 10104159001), in DMEM). Separated lung lobes were minced and incubated in enzyme solution at 37°C for 25-35 minutes. Dissociation was quenched with equivalent volume 10% FBS/DMEM and strained through a 100 µm strainer. Cell pellet was resuspended in red blood cell lysis buffer (100 µm EDTA, 10 mM KHCO3, 155 mM NH4Cl) for 2 minutes, followed by quenching with 10% FBS/DMEM and filtration through a 40 µm strainer. Cell pellet was resuspended in 500µL 2%FBS/DMEM and split into three fractions: CD45/31/140a, CD45, and bulk sample. For MACS, cells were incubated for 30 minutes with the following antibodies: CD45 (MiltenyiBiotech, 130-052-301), CD31 (MiltenyiBiotech, 130-097-418), and CD140a (MiltenyiBiotech, 130-101-547). MACS depletion was performed as per manufacturer protocol using LD columns (MiltenyiBiotech, 130-042-901). Lung cell suspensions were first enriched for epithelial and mesenchymal cell populations as follows: 11,200 cells from CD31/CD45/CD140a-depleted cells, 3200 cells from CD45-depleted cells, and 1600 cells from bulk fractions, and resuspended in PBS supplemented with 0.01% BSA. We used Drop-seq S3 (Second-Strand Synthesis), the method optimized by adopting S3 procedure from Seq-well S3 (Hughes et al., Immunity 2020 ). Briefly, cells at a concentration of 100 cells/μl were run through microfluidic channels at 3,000 μl/h, together with mRNA capture beads at 3,000 μl/h and droplet-generation oil at 13,000 μl/h. Captured mRNA on the beads were reverse-transcribed using Maxima H Minus RTase (Thermo Fisher, #EP0753) with a series of incubation at 25°C for 30 min, 42°C for 90 min and 50°C for 30 min followed by Exonuclease I treatment. The beads were treated with fresh 0.1M NaOH at room temperature for 5 min followed by second-strand synthesis using Klenow fragment (3'→5' exo minus, New England Biolabs, #M0212L) with incubation at 37°C for 60 min. DNA polymerase for The beads were then used for pre-amplification step (1 cycle of 985°C for 3 min, 4 cycles of 98°C for 15 sec, 55°C for 45 sec, 68°C for 4min, 10-1445–17 cycles of 98°C for 15 sec, 5765°C for 30 sec, 68°C for 4 min and 1 cycle of 72°C for 10 min) using was replaced by Terra PCR Direct Polymerase (#639271, Takara) which was ranked #1 polymerase in several studies (Sasagawa et al., Genome Biology 2013; Bagnoli et al., Nature Communications 2018). The rest of theother processes were performed as described in the original Drop-seq protocol. Drop-seq S3