GSM6341024: Astro_3_L2; Drosophila melanogaster; RNA-Seq
We dissociated a 50-100 µl pool of surface-sterilized Drosophila embryos (stage 17, 17-18.5 hours after egg laying) in C+G medium using a dounce homogenizer. Cells were then washed and filtered with 40 µm cell strainer. The concentration of viable cells was quantified with Trypan blue using a TC20 automated cell counter. Samples with a minimum viability of 80% were then diluted to 1000 cells/ µl in 1X PBS supplemented with .04% BSA and sequenced using the 10X NextGem v3.1 single cell platform. 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.