RNA-seq analysis of HTD114 cell nuclei

Nuclei were isolated from HTD114 cells following lysis in 0.5% NP40, 140 mM NaCl, 10 mM Tris–HCl (pH 7.4), and 1.5 mM MgCl₂. Nuclear RNA was isolated using TRIzol reagent using the manufacturer's instructions, followed by DNase treatment to remove possible genomic DNA contamination. RNA-seq was carried out at Novogene. Briefly, ribosomal RNAs were removed using the Ribo-Zero kit (Illumina), RNA was fragmented into 250–300 bp fragments, and cDNA libraries were prepared using the Directional RNA Library Prep Kit (NEB). Paired-end sequencing was done on a NovaSeq 6000. Triplicate samples were merged and aligned to the human genome (hg19) using the STAR aligner (Dobin et al. 2013 (link)) with default settings. Duplicate reads and reads with map quality below 30 were removed with SAMtools (Li et al. 2009 (link)).