GSM8174379: E18_OVA_scATACseq; Mus musculus; ATAC-seq
Lung tissues were digested in RPMI enzyme solution containing collagenase D (2 mg/ml), DNase I (0.1 mg/ml), and Dispase II (0.2 mg/ml, Roche 04942078001) at 37°C for 40 min (for adults) or 20 min (for fetuses). Digestion was performed by changing the solution every 20 min. And then, lung tissues were digested in RPMI enzyme solution containing collagenase D (2 mg/ml), DNase I (0.1 mg/ml), Dispase II (0.2 mg/ml) and Liberase TH (0.01 mg/ml, Roche 5401151001) at 37°C for 20 min. The digested samples were filtered through 70 µm strainers and suspended in 36% Percoll (Cytiva, 17-0891-01) diluted with RPMI, 72% Percoll diluted with PBS was injected into the bottom and centrifuged at 800 × g for 20 min at 25°C. Cells were collected in the interlayer between 36% and 72% Percoll, washed with RPMI supplemented with FBS (2% v/v), and stained for flow cytometry. In fetal lung cell collection, ILC2 (CD45+, Lin-, ST2+), immune cells (CD45+) and epithelial cells (EpCAM+) were purified using FACSMelody (BD Biosciences). The nuclei were isolated according to the protocol of “Nuclei Isolation from Embroynic Mouse Brain for Single Cell Multiome ATAC + Gene Expression Sequencing, Protocol 1” (10x Genomics, CG000366). Briefly, cells were centrifuged at 500 × g for  5 min at 4°C and removed the supernatant. The pellets were added 100 µl chilled Lysis Buffer (1 mM Tris-HCl (pH 7.4), 1 mM NaCl, 0.3 mM MgCl2, 0.01% Tween-20, 0.01% Nonidet P40 Substitute, 0.001% Digitonin, 0.1% BSA, 0.1 mM DTT, 0.1 U/µl RNase inhibitor), and incubated for 5 min on ice. The lysed cells were washed by 1ml chilled Wash Buffer three times. Libraries were prepared according to the protocol of Chromuim Next GEM Single Cell Multiome ATAC + Gene Expression Kit using a Chromium controller (10x Genomics). Briefly, GEMs were first generated by Master Mix with barcoded Single Cell Multiome Gel Beads (10x Genomics, PN-1000285), neuclei and distribution to Oil to Chromium Next GEM Chip J (PN-1000230). Barcoded cDNA was then generated from the GEM. This cDNA was amplified by PCR reaction to generate Gene Expression library and ATAC library (10x Genomics, PN-1000212, PN-1000215).