title: Comparative analysis of single-cell and spatial Nanopore long-read methods
design: Comparative analysis of single-cell and spatial Nanopore long-read methods
library_construction_protocol: As described in Lebrigand et al. 2020, we depleted the cDNA for variable extended (30–50%) cDNA that lacks poly(A) and poly(T) sequences. We re-amplified 10 ng of the 10x Genomics PCR product for  5 cycles with 5′-NNNAAGCAGTGGTATCAACGCAGAGTACAT-3′ and 5′ Biotine-AAAAACTACACGACGCTCTTCCGATCT 3′. After 0.6x SPRIselect purification to remove excess biotinylated primers, biotinylated cDNA (in 40 μl EB) is bound to 15 μl 1x SSPE washed Dynabeads™ M-270 Streptavidin beads (Thermo) resuspended in 10 μl 5x SSPE for 15 min at room temperature on a shaker. After two washes with 100 μl 1x SSPE and one wash with 100 μl EB, the beads are suspended in 100 μl 1x PCR mix and amplified for 9 cycles with the primers NNNAAGCAGTGGTATCAACGCAGAGTACAT and NNNCTACACGACGCTCTTCCGATCT to generate enough material for Nanopore sequencing library preparation. Amplified cDNA was purified with 0.6x SPRISelect  and Nanopore sequencing libraries were prepared with the Oxford Nanopore SQK- LSK-110[MOU2]  kit following the manufacturer's instructions. All PCR amplifications for Nanopore library preparations were done with KapaHifi Hotstart polymerase (Roche Sequencing Solutions): initial denaturation, 3 min at 95 °C; cycles: 98 °C for 30 s, 64 °C for 30 s, 72 °C for 5 min; final elongation: 72 °C for 10 min, primer concentration was 1 μM.