Dnase 1

For GNPs isolation, we followed the previously described protocol (Li et al., 2013 (link)), cerebella were harvested from C57BL/6J mice at P4-P7 then digested in a solution containing 10 units/ml papain (Worthington), 25 U/ml DNase I (Solarbio) and 2 mg L-cysteine (Solarbio) at 37°C for 30 min to obtain a single-cell suspension. After filtered with 70 μm strainer, cells were centrifuged through a 35%–65% Percoll gradient (GE Healthcare). Cells from the 35–65% interface were suspended in Neurobasal Medium with B27 supplement. Then GNPs were suspended in NB-B27 and plated on Poly-L-omithine hydrobromide (PLO) and Laminin (all from Sigma)-coated coverslips. GNPs was treated with 5 μM PTL, 50% sonic hedgehog conditional medium (Shh-CM) and Shh-CM adding 5 μM PTL for 24 h. EDU (Proteintech) was added 20 μM and incubated GNPs for 1 h. Then stained according to the instructions.

The murine lungs were cut into fine pieces and digested with 1 mL of tissue digestive fluid containing 0.3 mg/mL collagenase IV (Sigma‒Aldrich, St.145 Louis, MO, USA), 25 U/mL DNAse I (Solarbio Life Science, Beijing, China) and 5% foetal bovine serum (FBS) in RPMI (Biological Industries, Kibbutz Beit Haemek, Israel) for 30 min at 37 °C and filtered through a 70 μm nylon filter. The red blood cells were then further removed from the isolated cells using 2 mL/lung Red Blood Cell Lysis Buffer (Solarbio Life Science). All isolated cells were resuspended in staining buffer. The procedures for single-cell isolation from the piglet lung were similar to those from the mouse lung. A piece of the lung from the lesions was cut into small pieces and added to 5 mL of digestive solution. After following the steps in mouse cell isolation to obtain single cells, these cells were further centrifuged using OptiPrep separation medium (Axis-Shield, Dundee, UK) to remove dead cells. The isolated cells were immediately used in RNA-seq experiments.

Total RNA was extracted from protonemata of P. patens using a RNeasy Plant Mini Kit (Qiagen) and DNase I (Solarbio). cDNA was synthesized using M-MLV reverse transcriptase (Promega M1701). qRT–PCR was performed using Bio-Rad CFX96 Real-Time System (Thermo Fisher Scientific) and TransStart Top Green qRT-PCR kit (Transgen Biotech), with three independent biological replicates. PpEF1α (elongation factor 1-alpha, Phypa_439314) was used as reference gene to calculate the relative expression.

Cells from intestinal lamina propria, Peyer’s patches, and mesenteric lymph nodes were isolated as described previously (Rios et al., 2016 (link)). Briefly, Peyer’s patches and mesenteric lymph nodes were mechanically dissociated in ice-cold PBS. The resulting cell suspensions were passed through a 70-um mesh cell strainer. To prepare the intestinal lamina propria cells, associated fat and Peyer’s patches were removed, the intestinal tissue was washed in ice-cold PBS to remove the luminal contents and cut open longitudinally, and the tissue was cut into four equal-sized pieces. Epithelial cells were removed by shaking the tissues in PBS with 1 mM EDTA, 1 mM dithiothreitol, and 10% fetal calf serum for two rounds of 20 min at 37°C. Then, the pieces were washed three times with PBS to remove the EDTA, minced exactly 40 times in a microfuge tube, and incubated in 20 ml of RPMI-1640 supplemented with 1.5 mg ml^-1 Collagenase II (Biosharp), 2.5 mg ml^-1 hyaluronidase (Biosharp), and 0.25 mg ml^-1 DNase I (Solarbio) for 45 min at 37°C with constant shaking. Cell suspension was then extracted by passing the tissue and supernatant over a 70-µm mesh cell strainer. The cell suspension was then centrifuged, and the resuspended pellet was further purified from the interface of a 45/72% Percoll density gradient.

Cells from colonic lamina propria (LP), mesenteric lymph nodes (MLN) and spleen were isolated as described previously [22 (link), 27 (link)]. Briefly, MLN and spleen were mechanically dissociated in ice-cold PBS. The resulting cell suspensions were passed through a 70-μm mesh cell strainer, and spleen also needed erythrocyte lysis. To prepare colonic LP cells, associated fat and Peyer’s patches were removed, the colonic tissue was washed in cold PBS to remove fecal contents and cut open longitudinally, and the colon was cut into 2-cm pieces and washed twice in 2 mL PBS containing 1 mM dithiothreitol, 10% FCS and 1 mM EDTA with constant agitation for 40 min at 37 °C. And then the pieces were minced exactly 50 times to obtain 1 mm tissue fragments and incubated in 2 mL of RPMI-1640 supplemented with 2.5 mg/mL Hyaluronidase (Biosharp), 1.5 mg/mL Collagenase II (Biosharp), and 0.25 mg/mL DNase I (Solarbio) with constant agitation for 45 min at 37 °C. The cells suspension was extracted through filtration and centrifugation, and the resuspended pellet was further purified from the interface of a 45%/72% Percoll density gradient.

Back skin tissues dissected from euthanized mice were placed into dishes and cut into 0.2mm² pieces with scissors. The pieces of tissue were transferred into 100 mm dishes and incubated with a digestion solution containing 2.5 mg/mL collagenase I (Cat # C0130, Sigma), 2.5 mg/mL trypsin (Cat # 27250018, Thermo Fisher Scientific), and 1 U/mL DNase I (D8071, Solarbio) in Roswell Park Memorial Institute (RPMI) 1640 medium for 2 h at 37°C. The digested supernatant was filtered through a 70 μm cell strainer to obtain a single-cell suspension. Skin lymphocytes were isolated by centrifugation in lymphocyte separation solution (Dakewe Biotech, Beijing, China). CD11c-positive dendritic cells were enriched with the EasySep Mouse CD11c Positive Selection Kit (Stemcell, Cat# 18780) for subtype sorting.