GSM9659756: hPCO_D22_ipsa; Homo sapiens; RNA-Seq
For single-nucleus RNA sequencing (snRNA-seq), hPCOs at day 22 derived from H1 and IPSA cell linesof differentiation were collected and prepared for sequencing. Organoids were pooled together and immediately cryopreserved at -80°C (with the culture medium carefully removed before freezing). Nuclear isolation was performed using the Chromium Nuclei Isolation Kit with RNase Inhibitor following the manufacturer's protocol. In brief, the frozen organoids were mechanically disrupted using a pestle in Lysis Buffer and filtered through a specialized column. Cellular debris was then eliminated by centrifugation in Debris Removal Buffer. The purified nuclei were washed, resuspended, and processed to construct cDNA libraries using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index). Specifically, the nuclear suspension was compartmentalized into nanoliter-volume Gel Bead-In-Emulsions (GEMs). The nuclear suspension was introduced at limiting dilution into a microfluidic channel containing Single Cell 3' v3.1 Gel Beads, followed by oil phase introduction. Following cell lysis, an Illumina TruSeq Read 1 sequence, a 16-nucleotide 10x Barcode, a 12-base pair unique molecular identifier (UMI), a 30-nucleotide poly(dT) sequence, and a Capture Sequence 1 were released into the lysate containing the reverse transcription master mix, enabling synthesis of full-length cDNA from polyadenylated RNA. After droplet disruption and cleanup of non-cDNA components using silane-coated magnetic beads, the libraries underwent size selection. TruSeq Read 2, P5, P7, i7, and i5 sample index sequences were then appended to each size-selected cDNA molecule. Finally, the completed libraries were sequenced on the Illumina Novaseq6000 platform using a configuration of 28bp for read1 and 151bp for read2.