GSM9157988: WT_P28_808; Mus musculus; RNA-Seq
Lungs from postnatal day 5 and 28 mouse pups were collected and digested with Collagenase I tissue removal solution (Collagenase/Dispase (2.5 mg/ml), and DNase (30 μg/ml) in PBS) at 37ºC for 60 minutes with brief vortexing every 10-15 minutes. Single cells were passed through a 40um cell strainer in PBS containing 0.04% BSA. 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 loade 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 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 appropriate sized fragments were selected with SPRiselect reagent for subsequent library construction.During the library construction llumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.