GSM8174393: E18_Lung_scRNAseq_Tag; Mus musculus; RNA-Seq
For the preparation of lung immune cells for flow cytometry analysis and scRNA-seq, lung tissue was digested in RPMI enzyme solution containing collagenase D (2 mg/ml, Roche, 11088882001) and DNase I (0.1 mg/ml, Roche, 10104159001) at 37°C for 60 min (for adults) or 40 min (for fetuses). Digestion was performed by changing the solution every 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. The tags were assigned as follows: C0301, fetal lung from DEX-treated mother 1; C0302, fetal lung from DEX-treated mother 2; C0307, fetal lung from PBS-treated mother 1; C0308, fetal lung from PBS-treated mother 2. ILC2s (FVD-CD45+Lin- ST2+) were purified by FACSMelody (BD Biosciences). Libraries were prepared according to the protocol of Chromuim Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) (10x Genomics, PN-1000263) using a Chromium controller (10x Genomics). Briefly, GEMs were first generated by Master Mix with barcoded Single Cell VDJ 5' Gel Beads, cells and distribution to Oil to Chromium Next GEM Chip K (10x Genomics, PN-1000286). Barcoded cDNA was then generated from the GEM. This cDNA was amplified by PCR reaction to generate, 5' Gene Expression library and Feature Barcode library (10x Genomics, PN-1000190, -1000256, -1000215, and -1000250). Sequencing was then performed by Novaseq 6000 (Illumina).