GSM8174377: E18_OVA_snRNAseq; Mus musculus; RNA-Seq
Fetal 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 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). snRNA-seq