Nanopore sequencing of telomerase products
293T TERC-null cells were transfected with a 5:1 ratio of ransfections were performed using Lipofectamine LTX with Plus Reagent (Invitrogen) in Opti-Mem (Gibco) according to the manufacturers instructions. On the evening prior to transfection, two million cells were plated per well of a six well dish, then transfected with 2.5 g total DNA per well. pBS U3 TERC 500 modified to express AAAGGG encoding TERC was co-transfected with PCDNA3.1 3XHA TERT in a 5:1 mass ratio. Two days after transfection, cell extracts for TRAP assays were generated made using TRAPeze1X CHAPS Lysis Buffer (Roche) supplemented with RNASEin (Promega). Telomerase extracts were added to reactions AAAGGG TRAP primers and dNTPs were incubated for 30 minutes at 30C followed by heat inactivation at 95C for 5 minutes, and purification of oligonucleotides using the oligo clean and concentrator kit (Zymo) and elution in 15ul of water. 10 ul of elutate was added to 50ul reactions containing TRAP buffer, taq and 50uM of each dNTP, and PCR was performed. T-free TRAP products were prepared for sequencing using the Ligation Sequencing Kit with the Native Barcoding Expansion (Oxford Nanopore Technologies SQK-LSK109, EXP-NBD104) according to the manufacturers instructions, except that a ratio of 4:1 of SPRI Purification Beads (Beckman Coulter) were used for all purification steps. Samples were pooled after barcode ligation, adapters were ligated using per the manufacturers instructions, then products were loaded onto a Flongle flow cell (FLO-FLG001). Reads were first analyzed using the high accuracy mode (Guppy) and then reads with Q score >9 were re-base called using super-high accuracy mode (Guppy), and reads which had Q score>10 were used for further analysis. MinKNOW 22.05.5, Bream 7.1.3, Configuration 5.1.5, Guppy 6.1.5, MinKNOW Core 5.1.0