RACE-Nano-Seq of etoposide treated K562
The human chronic myelogenous leukemia cell line K562 was treated with 90 M etoposide (Abmole), a topoisomerase II inhibitor, for 36 hours to induce DNA damage. Total RNA was extracted using TRNzol Universal (Tiangen, DP424) and the Total RNA Kit I (Omega, R6438-02). PolyA+ RNA was isolated with mRNA capture beads (VAHTS, N401) and subjected to 5 and 3 Rapid Amplification of cDNA Ends (RACE) to enrich for full-length transcripts, targeting 12 anchor exons across 4 genomic loci. The coordinates of the anchor exons in the human GRCh38/hg38 reference genome are as follows: chr1:22264739-22264838(), chr1:22271593-22271687(), chr1:22283283-22283382(), chr1:22285184-22285367(), chr12:1497926-1498123(+), chr12:1500043-1500235(+), chr12:1509757-1511057(+), chr20:23159772-23159868(+), chr20:23178710-23178772(+), chr7:132076399-132076891(), chr7:132077817-132077939(), and chr7:132080666-132080857(. Separate reactions were conducted for the 5' RACE and 3' RACE for each anchor exon essentially as described in our previous publication (PMID: 38001496). The 5' RACE and 3' RACE products from each reaction were mixed in equal volumes and purified with 2 volumes of VAHTS DNA Clean Beads (Vazyme) to a final volume of 50 L. The concentrations were measured using Merinton SMA6000 spectrophotometer. The pooled products of 5' RACE and 3' RACE were sequenced using Oxford Nanopore Technologies platform by BENAGEN Corporation (Wuhan, China). The sequencing library was prepared with the Ligation Sequencing Kit (SQK-PCS109) from Oxford Nanopore Technologies Inc. (Oxford, UK). Sequencing was performed on a FLO-PRO002 R10.4 flow cell using Oxford Nanopore PromethION (Oxford Nanopore Technologies Inc., Oxford, UK). A total of 23.2 GB of raw data were obtained after 48 hours of sequencing. The reads were based-called in real-time using MinKNOW (v20.10.6) and integrated with Guppy (v4.2.3). Raw Nanopore sequencing data were filtered to retain sequences with an average quality score of 7 or higher, resulting in 18.0 GB of clean data.