GSM4785406: 2_Rtkn2_AT1_C; Mus musculus; ATAC-seq
Perfused lungs were collected from anesthetized mice as described above. Previously published protocols for cell dissociation and sorting cells were used with minor modifications11. Extra-pulmonary airways and connective tissues were removed from lungs, which were then minced with forceps and digested in Liebovitz media (Gibco, 21083-027) for scRNA-seq, ATAC-seq, or scATAC-seq with 2 mg/mL collagenase type I (Worthington, CLS-1, LS004197), 0.5 mg/mL DNase I (Worthington, D, LS002007), and 2 mg/mL elastase (Worthingon, ESL, LS002294) for 30 min at 37 °C. To stop the digestion, fetal bovine serum (FBS, Invitrogen, 10082-139) was added to a final concentration of 20%. Tissues were triturated until homogenous and filtered through a 70 µm cell strainer (Falcon, 352350) on ice in a 4 °C cold room and transferred to a 2 mL tube. The sample was then centrifuged at 1,537 rcf for 1 min; then 1 mL red blood cell lysis buffer (15 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA, pH 8.0) was added and incubated for 3 min before centrifugation again at 1,537 rcf. The red blood cell lysis buffer incubation was repeated to remove residual red blood cells. Liebovitz+10% FBS was used to wash and resuspend samples, which were then filtrated through a 35 mm cell strainer into a 5 mL glass tube and had SYTOX Blue (1:1000, Invitrogen, S34857) added. After refiltering, samples were for ATAC-seq were sorted for GFP positive cells from a RosaSun1GFP reporter activated by Wnt3aCre, Rtkn2CreER, or SftpcCreER on a BD FACSAria IIIu cell sorter with a 70 mm nozzle. Samples for scRNA-seq or scATAC-seq were stained with CD45-PE/Cy7 (BioLegend, 103114), ECAD-PE (BioLegend, 147304), and ICAM2-A647 (Invitrogen, A15452) antibodies (1:250 dilutions for all antibodies) for 30 min followed by a wash and being resuspended with Liebovitz meda+10% FBS and addition of SYTOX Blue. Samples were then filtered through a 35 mm cell strainer into a 5 mL glass tube again and sorted by a BD FACSAria Fusion sorter or a with a 70 mm nozzle. The OMNI-ATAC protocol68 was followed with minor modifications. 60,000-100,000 sorted GFP cells were centrifuged at 384 rcf for 5 min at 4 °C. The cell pellet was then resuspended by pipetting three times in 50 µl of cold ATAC-RSB lysis buffer (0.1% NP-40, 0.1% Tween, 0.01% Digitonin, 10 mM Tris-HCl pH 8.1, 10 mM NaCl, 3 mM MgCl2) and incubated for 3 min. Then 1 mL of cold ATAC-RSB + Tween (10 mM Tris-HCl pH 8.1, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween) was added. The sample tube was then inverted three times and centrifuged at 384 rcf for 10 min at 4 °C. After discarding the supernatant, the pellet was resuspended in transposition mixture composed of 22.5 µl of Reaction Mix (PBS, 2.2% Digitonin, 2.2% Tween 20), 25 µl of transposase buffer (10 mM MgCl2, 20mM Tris- HCl, 20% Dimethyl Formamide), and 2.5 µl of Tn5 enzyme (NX#-TDE1, Tagment DNA Enzyme,15027865, Illumina) and incubated at 37 °C for 30 min in a thermocycler. Samples were then purified using the MinElute PCR Purification kit (Qiagen, 29004) and eluted with 10 µL of H2O. Amplification and barcoding of ATAC libraries was carried out using the Greenleaf primers for 12 cycles of PCR enrichment following the standard OMNI-ATAC amplification PCR program. A volume of 5 µL was taken after amplification to examine amplification efficiency prior to size selection. A double-sided size selection was then performed (.5 x -1.8 x volume) using the SPRIselect reagent (Beckman Coulter, B23318). Samples were then verified for library size and absence of primer dimers by gel electrophoresis and concentration was measured using the Qubit HS dsDNA assay (ThermoFisher Scientific, Q32851).