GSM7047812: CD343; Mus musculus; RNA-Seq
Extracted lungs were infused with 1 ml digestion solution (dispase with 0.2 mg/ml DNase I). Lungs were incubated at room temperature for 45 min with gentle agitation and tissue was gently teased apart with forceps into 1-2 mm fragments and incubated in digestion buffer for 15 min. Solution was filtered through a 70 µm cell strainer, washed with DMEM containing 5% FBS, centrifuged, and RBCs lysed with ACK lysing buffer. Resulting single cell suspension was filtered twice through a 40 µm cell strainer with DMEM containing 5% FBS. Cells were counted after staining with 7-AAD for exclusion of nonviable cells, cell viability exceeded 95%. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction.