GSM8712996: Lung, Hgf mutant, P21; Mus musculus; RNA-Seq
Lung lobes were dissected, finely minced using sharp scissors and transferred into enzymatic digestion solution containing 40U/ml collagenase I (Gibco, 170100-017), 40U/ml Collagenase IV (Gibco, 170100-019), 4U/ml Dispase II (Thermo Fisher, 17105041) and 2 U/µl DNase I (Worthington, LK003170) prepared in DMEM (Gibco, 31053028) medium. Digestion was performed in a water bath, at 37°C for 30 min. During this time, the tissue was also mechanically dissociated by passing it through a needle-syringe for several times.  Enzymatic digestion was stopped by addition of FACS buffer (2% FBS in DMEM) and the lysate was passed through 70 µm cell strainer (Falcon, 352350). The filtrate was centrifuged for 5 min at 4°C and the pellet was resuspended in RBC lysis solution (eBioscience, 00-4333-57) and further incubated for 10 min on ice. Post resuspension in FACS buffer, the cell lysis was pass through a 50 um Cell Trics filter (Sysmex, 04-0042-2317). Cells were centrifuged for 5 min at 4°C and the pellet was resuspended in cold MACS depletion buffer (autoMACS Running Buffer-MACS Separation Buffer, Miltenyi Biotec, 130-091-221). To proceed with magnetic separation and depletion of CD45+ cells and erythrocytes from the cell suspension, CD45 microbeads (Miltenyi Biotec, 130-052-301) and Ter119 microbeads (Miltenyi Biotec, 130-049-901) were added to the cells resuspended in MACS depletion buffer and incubated for 20 min at 4°C. After washing, the cell solutions were passed through MACS MS column (Miltenyi Biotec, 130-042-201) attached to a MACS separator placed on a magnetic Multistand. Filtrate solution consisting of CD45-/Ter119- cells was collected and cell number was counted using Luna Automated Cell Counter (L10001). For each mouse genotype, 40000 cells were prepared for further sequencing from the two mouse littermates, using equal cell number of cells per sample. For the single cell sequencing of postnatal cerebral cortex, two P12 Nodal lox/lox  control and two Nodal iPCKO mice were anesthetized by intraperitoneal injection of Xylazine (16mg/Kg) and Ketamine (100mg/Kg) and transcardially perfused with 10 mL of ice-cold PBS supplemented with Heparin (25 U/mL). Brains were collected and the meningeal layers removed. Next, the cerebral cortex of each hemisphere was dissected and transferred to ice-cold DMEM supplemented with Penicillin/Streptomycin, Glutamine (GlutaMAX, Gibco) and 25 mM HEPES (PAA, Cat. No. S11-001), hereafter dissection media. The cortices belonging to mice from the same genotype were pooled together and minced using scalpels. The resulting paste was resuspended in 1 mL of enzyme blend containing Papain (25 U/mL, Worthington Cat. No. LK003176), DNAseI (113 U/mL, Worthington Cat. No. LK003170), and Liberase DH (100 µg/mL, Roche Cat. No. 5401054) dissolved in pre-warmed dissection media and incubated 30 min at 37oC. During this time, the tissue was further homogenized by repeatedly pipetting the solution up and down every 10 minutes using filtered 1mL tips. After the first 15 min of incubation, 0.5 mL of the described enzyme blend were further added. Next, the tissue homogenate was filtered through a 70µm nylon mesh into a 50mL Falcon tube, the filter was washed with 1 mL of pre-warmed dissection media and the final volume obtained was measured. In order to remove debris and myelin, the cell suspension was mixed with 1.7x volume 22% BSA (Carl Roth Cat. No. 8076.2) dissolved in PBS and centrifuged at 1000 g for 12 min and RT. The supernatant was aspirated and the remaining cell pellet resuspended in 1mL of the described enzyme blend for a final 20min incubation step at 37oC with pipetting every 5min. The resulting single-cell suspension was filtered through a 40µm cell strainer and diluted by addition of 8mL of pre-warmed dissection media. After centrifugation at 300 g for 5min, the supernatant was discarded and the cell pellet resuspended in 1ml of Red Blood Cell Lysis Buffer (Sigma Cat. No. R7757). After a 1 min incubation at RT, 20ml of ice-cold PBS supplemented with 2% fetal calf serum were added and the whole suspension centrifuged at 300 g for 5 min at 4oC. Next, the supernatant was discarded and the cell pellet resuspended in 1ml of filter-sterilized endothelial cell buffer (15 mM HEPES, 153 mM NaCl, 5.6 mM KCl, 1.7 mM CaCl2, 1.2 mM MgCl2 and 10% BSA, pH 7.4) (PMID: 30637296). Cell concentration was assessed using an automated cell counter and 4 x 104 cells from each genotype were loaded into BD Rhapsody Cartridge (BD Biosciences Cat. No. 633733) for cell capture. Cells were loaded into a BD Rhapsody Cartridge (BD Bioscience, 633733) and captured on the BD Rhapsody Express Single-Cell Analysis System (BD Bioscience). Single-cell mRNA whole transcriptome (WTA) libraries were created using the BD Rhapsody Whole Transcriptome Analysis (WTA) Amplification Kit (BD Bioscience, 633801) and DNA sequencing was performed on a NextSeq500 (Illumina) using 2 x 75 bp paired end reads with an 8 bp single index.