GSM5503580: p14_BPD_saline; Mus musculus; RNA-Seq
The lungs obtained from two pups in each group were analyzed for scRNA-seq. The lungs obtained for scRNA-seq  were rinsed with Dulbecco's PBS (Nacalai, kyoto, Japan).followed by ice-cold 10%FBS-RPMI (Nacalai, kyoto, Japan). Then the tissues were minced in a Petri dish on ice.  Lung pieces were transferred to a GentleMACS C tubes (Miltenyi Biotec, Bergisch Gladbach, Germany), using an enzyme mix containing dispase (50 caseinolytic units/ml, Corning, New York, USA), collagenase (2mg/ml, Wako, Osaka, Japan), elastase (1mg/ml, Worthington Biochemical Corporation, New Jersey, USA), and DNase (10unit/ml, Takara, Shiga, Japan). Samples were homogenized using a GentleMACS Dissociators (Miltenyi Biotec, Bergisch Gladbach, Germany), filtered using a 70 μm strainer, and pelleted by centrifugation at 300 g for 5 min, resuspended in ice-cold RBC lysis buffer (Bio Legend; 420301) for red blood cell lysis, and then counted. Live cells were sorted and collected using FACS AriaⅢ (Becton Dickinson, New Jersey, USA). The cells were washed twice with PBS with 0.1% BSA and suspended to 1x106cells/ml. Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1(10x Genomics, California, USA) was used to prepare the library according to the manufacturer's protocol. Cells were loaded to a target of 6,000 cells.  The sequencing libraries were sequenced on the NextSeq 2000 platform using NextSeq 2000 P3 Reagents (100 Cycles) (illumine, California, USA). The read length was set to 28 (read 1) + 8 (i7) + 0 (i5) + 91 (read 2) bases.