GSM7661370: Line#2, D6, tam-inducible, scRNAseq, rep2; Homo sapiens; RNA-Seq
Stem cells / Differentiated EECs were dissociated using 1x TrypLE Exprress at 37 °C for 10 min. Cells were washed and re-suspended in PBS and stained with a Viability Dye, washed and resuspended in FACS buffer (HBSS without Ca+2, Mg+2 and phenol red, 0.9% Glucose, 10 mM HEPES, 500 ppm N-acetyl L-cystein, 10µM Y27632,  2% FBS). Alive cells were sorted using a FACS (BD Aria) according to the presence or absence of mCherry signal. Cells were washed and resuspended in PBS supplemented with 0.04% w/v BSA. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells 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. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.