GSM8956372: D4_351KO; Mus musculus; RNA-Seq
Mouse lungs were perfused with 4 U/ml elastase (Worthington Biochemical Corporation, NJ) and digest with 100 U/ml DNase I (Sigma), and resuspended in Hanks' balanced saline solution supplemented with 2% fetal bovine serum (FBS), 10 mM HEPES, 0.1 mM EDTA (HBSS+ buffer). The cell suspension was incubated with primary antibodies including CD24-PE-Cy7, EpCAM-Percp-Cy5.5, Sca-1-PE-Cy7, biotinylated-CD31, -CD34, and -CD45 for 45 minutes. Biotin-conjugated antibodies were detected following incubation with streptavidin-APC-Cy7 (catalog # 405208, BioLegend) for 30 minutes. Dead cells were discriminated by Fixable Viability Dye eFluor™ 780 (eBioscience) staining. Library was perfoemed according to the manufacter's instrutions (Single cell 3' Reagent Kits v3.1 protocol, 10x Genomics).Briefly, the cell suspension was resuspended in the master mix and loaded together with partitionling oil and gel beads into Chromium microfluidic chips to genernate the gel beads-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an llumina 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. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification. Sequencing was performed with NovaSeq PE150 according to the manufacturer's instructions (Illumina).