RNA-seq of U2OS cells treated with doxycycline-induced shRNAs targeting lamin A, LMNA and SYNE2

To create doxycycline-inducible shRNA knockdown stable cell lines, shRNAs targeting scramble, lamin A, LMNA, and SYNE2 were cloned into pLKO-Tet-On vectors and transfected into HEK293T cells alongside helper plasmids to produce lentiviral particles. U2OS cells were infected, selected with puromycin, and treated with doxycycline to induce gene-specific knockdown. Total RNA was extracted, ribosomal RNA removed, and RNA libraries prepared for sequencing using standard protocols, including cDNA synthesis, adapter ligation, and PCR amplification. Library quality was verified, and sequencing was performed on an Illumina NovaSeq 6000 to generate 150 bp paired-end reads.