Nanopore RCA long-read RNA-seq of PC3E and GS689 cell mixture
Single-cell cDNA was generated using the Chromium Next GEM Single Cell 3 Kit v3.1 and Chip G Single Cell Kit (10x Genomics). The GEM-RT incubation time was extended to 90 min, and the initial PCR elongation time was extended to 4 min, with 13 PCR cycles performed. Approximately 510 ng of the initial PCR product was re-amplified for eight cycles using 10x partial TSO and 10x partial read 1 primers. Amplified cDNA was purified with SPRISelect beads (0.7 volume).Circularization of cDNA was performed using the Gibson Assembly reaction with a DNA splint, followed by digestion of linear DNA with exonucleases and purification using SPRISelect beads. RCA was conducted with Phi29 DNA polymerase at 30C for 16 hours, and a debranching reaction was performed to mitigate higher structure formation. DNA size was monitored using a TapeStation, targeting a predominant peak around 89 kbp. Debranched RCA products were size-selected using AMPure XP beads and long-fragment washing buffer to remove fragments under 3 kbp.Nanopore 1D cDNA libraries were prepared using the Native Barcoding Kit (SQK-NBD114.24) with Long Fragment Buffer for purification. Final libraries were loaded onto R10.4.1 flow cells and sequenced on PromethION with a minimum read length of 1,000 bp.