GSM7661361: Line#2, D3, tam-inducible, scATACseq; Homo sapiens; ATAC-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. Nuclei were extracted and resuspended in PBS supplemented with 0.04% w/v BSA. Library was performed according to the manufacter's instructions (single cell ATAC v1.1 protocol, 10x Genomics). Briefly, Transposed nuclei 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 transposed DNA elements contained in every single GEM was amplified, which contains an Ilumina P5 sequence and the 10x Barcode. The pooled barcoded ATAC sites 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 P7 sequences and a sample index were added.