AN003_RTCB_depletion_PARK7_NLS_SthCsm_targeting.fastq
Amplicons from RT-qPCR reactions were purified with magnetic beads (Mag-Bind TotalPure NGS), and ~13 ng of DNA was used to prepare sequencing libraries as described in SQK-LSK114 protocol using Native Barcoding Kit 24 V14 (SQK-NBD114.24) or Native Barcoding Kit 96 V14 (SQK-NBD114.96). Edited XIST RNA was reverse-transcribed using the LunaScript RT SuperMix Kit (NEB), and cDNA was used for PCR with primers XIST_seq_F and XIST_seq_R (table S7) and Q5 polymerase (NEB). Amplicons were purified with magnetic beads (Mag-Bind TotalPure NGS), and ~13 ng of DNA was used to prepare sequencing libraries as described in SQK-LSK114 protocol using Native Barcoding Kit 24 V14 (SQK-NBD114.24). ~20 fM of the library was loaded on the Nanopore MinION [R10.4.1 flow cell, accurate mode (260 bases per second)]. The flow cell was primed, and sequencing libraries were loaded according to the Oxford Nanopore protocol (SQK-LSK114). The sequencing run was performed in the fast base calling mode in the MinKNOW software. After the run, raw sequencing data (POD5 files) was re-basecalled with the super-accuracy model (dna_r10.4.1_e8.2_260bps_sup@v4.1.0) using and demultiplexed with Dorado basecaller (v0.3.3, Oxford Nanopore).