title: long-read RNA-seq of human brain with FTD
design: Single-nucleus suspension was isolated from fresh-frozen human brain samples. After 10x Genomics library preparation, the first-round PCR protocol was performed by applying 12 cycles of linear/asymmetric amplification to enrich molecules containing a 10x barcode with primer Partial Read1. The second-round PCR was performed by applying 6 cycles of exponential amplification under the same conditions with forward primer Partial Read1 and reverse primer Partial TSO. Exome enrichment was applied to the cDNA purified from the previous step by using a customized probe set targeting exon junctions of ~3,600 genes of interest. For each sample, ~75 fmol cDNA underwent ONT library construction by using the Ligation Sequencing according to the manufacturers protocol. The ONT library was loaded on a PromethION Flow Cell and sequenced with a PromethION sequencer for 72 h. Base-calling was performed with Guppy by setting base-quality score >7.