GSM8869697: ALV organoid scRNA, G60; Homo sapiens; RNA-Seq
Lung organoids cultured under different stiffness conditions for 49 days were dissociated into single-cell suspensions using TrypLE (Thermo Fisher, Cat#12605028). The suspensions were passed through a 70 µm cell strainer (BD Falcon, Cat#352235) into FACS tubes, ensuring a single-cell yield of over 90%. Cell viability and concentration were assessed using Trypan Blue Stain (Invitrogen, Cat#15250061) with the Countess™ 3 Automated Cell Counter (Life Technologies). Final cell concentrations were adjusted to 1000 cells/µL with a viability of ≥80%. hALOs were sequenced on the 10x Genomics platform by Annoroad Gene Technology. Single-cell suspensions were combined with Gel Beads and oil droplets in separate channels of Chromium Chip G (10x Genomics, Cat#1000121) to generate Gel Bead-In-Emulsions (GEMs) via microfluidics. Following the manufacturer's protocol, mRNA was reverse-transcribed into complementary DNA (cDNA) and subsequently amplified into double-stranded cDNA. A-tailing was performed, followed by ligation of sequencing adapters, including the Read2 primer. The resulting libraries incorporated P5 and P7 adapters and dual indices, enabling compatibility with Illumina sequencing platforms. The quality-controlled libraries were sequenced on an Illumina platform by Annoroad Gene Technology, ensuring high-quality data acquisition for downstream analysis.