GSM8004904: Lineage traced early postnatal a-SMA+ lung cells; Mus musculus; RNA-Seq
Five Acta2-CreERT2, ROSA26R(Zs/+)mice were injected with 4-OH-TM (200 µg per day) at P6.5, P8.5 and P10.5. At P60.5, lungs were collected, minced and incubated at 37°C for 40 min in the enzyme mixture from Miltenyi Biotec Lung Dissociation Kit. During this incubation, tissue was subjected twice to gentle lung dissociation protocol in a gentleMACS dissociator (Miltenyi Biotec). After inhibiting enzymatic activity with 10% FBS, single cell suspensions from each mouse were pooled together and passed through a 100 µm cell strainer and centrifuged at 700 × gfor 10 min at 4°C. The pellet was washed in 1X PBS with 1% FBS, DAPI (nuclear stain) added to the cell suspension, and Zs+DAPI- cells were isolated by FACS. Trypan blue was used to aid in cell counting. The cells were pooled together in 0.04% bovine serum albumin in PBS and proceeded to library preparation and DropSeq. Single cell suspension in RT Master Mix (75 μl) was loaded on the Single Cell A Chip     and partitioned with a pool of ~750,000 barcoded gel beads to form nanoliter-scale Gel Beads-    In-Emulsions (GEMs). Each gel bead has primers containing: (i) an Illumina R1 sequence (read    1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI),    and (iv) a poly-dT primer sequence. Upon dissolution of the Gel Beads in a GEM, the primers        were released and mixed and incubated with cell lysate and RT Master Mix, producing barcoded,    full-length cDNA from poly-adenylated mRNA.