title: GSM9253579: Human lung organoid, period 7; Homo sapiens; RNA-Seq
library_construction_protocol: Alveolar organoids were differentiated from H9 hESCs in a stepwise protocol. Single-cell RNA-Seq libraries were prepared using SeekOne® Single Cell Whole Trancriptome Kit according to manufacturer's instructions (SeekGene Catalog No.K00801). Briefly, an appropriate number of cells were mixed with reverse transcription reagents and added to the sample wells of the SeekOne® DD Chip S3 (Chip S3). Then, Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in Chip S3. Subsequently, Cell-containing reverse transcription reagents and BHBs were encapsulated into emulsion droplets using SeekOne® Digital Droplet System. Immediately following transferring emulsion droplets into PCR tubes, fifteen cycles of annealing (ramping from 8 °C to 42 °C) followed by a 5-min heat inactivation at 85°C were performed to obtain barcoded cDNA. Next, the barcoded cDNA was purified from broken droplet and then twice PCR reactions were performed to remove the majority of ribosomal and mitochondrial cDNA. AMPure beads were used to purify cDNA from the post PCR reaction mixture. Finally, one forth volume of cDNA was fragmented, end repaired, A-tailed and ligated into sequencing adaptor. DNA amplified by index PCR contains any part of polyA or non-PolyA RNA as well as Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were purified using AMPure beads and quantified by quantitative PCR (KAPA Biosystems KK4824). The libraries were then sequenced on Illumina NovaSeq 6000 with PE150 read length or DNBSEQ-T7 platform with PE150 read length.