title: Normal bladder 5 (Nanopore)
design: For cell lines RNA was extracted using the RNeasy kit (Qiagen), and for the clinical samples the truXTRAC FFPE kit was used (Covaris). DNase treatment was performed using the Turbo DNase kit (ThermoFisher). cDNA was generated with SuperScript III (ThermoFisher) and polyT primers. TAKARA DNA polymerase (TAKARA) was used for PCR reactions. The entire UTX ORF was amplified using primers targeting 5'-UTR and 3'-UTR (Table S3) and analyzed by ethidium bromide staining after separation using a 1% agarose gel. The ASR region was amplified using primers targeting exon 12 and exon 17 of UTX. Observed bands were gel eluted, reamplified, cleaned up and subjected to Sanger sequencing. The primers also included an extension of Nanopore universal tags: 5-TTTCTGTTGGTGCTGATATTGC-[project-specific forward primer sequence]-3 5-ACTTGCCTGTCGCTCTATCTTC-[project-specific reverse primer sequence]-3. For Nanopore sequencing, PCR products were further processed following the manufacturer's instructions (PCR barcoding 96 amplicons SQK-LSK109). 0.5 nM of the first-round PCR was further amplified using the Nanopore Barcoding primers (EXP-PBC096). Thereafter, the barcoded amplicons were pooled, and a 0.75x AMPure bead clean-up (A63880, Beckman Coulter) performed to deplete unwanted fragments below 150 bp. Thereafter, the pool was subjected to NEBNext FFPE DNA Repair and Ultra II End-prep kits (M6630 and E7546, New England Biolabs). After that, Nanopore adaptors were ligated using NEBNext Quick T4 DNA ligase (E6056, New England Biolabs) and subsequently cleaned with AMPure XP beads (A63880, Beckman Coulter) using Nanopores short fragment buffer for washing the beads