Nanopore scCirRL-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). Approximately 100 ng of re-amplified cDNA product was used for circularization and exonuclease digestion. Circular dsDNA was purified with 80% SPRISelect beads and PCR amplified for 45 cycles using splint-for and splint-rev primers. The resulting PCR products were purified with 80% SPRISelect beads. Nanopore 1D cDNA libraries were prepared using the Native Barcoding Kit (SQK-NBD114.24) as per the manufacturers instructions. Approximately 200 ng of cDNA underwent end repair and dA-tailing with the NEBNext Ultra II End Repair/dA-Tailing Module, followed by purification with Short Fragment Buffer. Final libraries were loaded onto R10.4.1 flow cells and sequenced on PromethION.