GSM7661388: Line#2, D6 ASCL1-KO, tam-inducible, Multiome-seq, RNA; 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. Nuclei were extracted and resuspended in PBS supplemented with 0.04% w/v BSA. Library was prepared using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics). Cellular mRNA and ATAC sites were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics) and with specific primers amplifying the ATAC sites. SPRI bead size selection was performed in order to separate both the mRNA-derived cDNA (>300bp) and the ATAC sites (>180bp). For the mRNA derived cDNA and ATAC library preparation, we further proceeded according to the manufacturer's instructions for Next GEM Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics).