GSM8679241: Human vaginal epithelial organoids, E2 and P4 treated; Homo sapiens; RNA-Seq
The hVEOs were transferred to a digestion medium (1×trypLE) at 37°C for 5-8 min (depending on organoid size). For further physical digestion to isolate single cells, the  hVEOs were pipetted gently and carefully with two or three caliber glass pipettes with different inner diameters. Single cells were transferred to cold phosphate-buffered saline (PBS, with 0.1-1% HSA) drops. Finally, individual cells were picked into a lysis medium on ice with a mouth pipette. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, hVEOs 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 sinale GEM was retrotranscripted to cDNA, which contains an llumina R1 primer sequence, Unique Molecular ldentifier (UMl) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DvnaBeads. amplified by PCR and the apropiated sized fragments were selected with SPRlselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.