Strand-Specific RNA-Seq Library Preparation

Total RNA was extracted from U2932 cells using RNeasy Mini Kit (QIAGEN, #74104) and polyA+ transcripts isolated with oligo (dT)₂₅-conjugated magnetic Dynabeads (Thermo Fisher). Strand specific RNA-seq libraries were prepared following a published protocol^{62 (link)}. Briefly, RNA was chemically fragmented in first strand buffer, converted to cDNA using SuperScript® III reverse transcriptase (Invitrogen), end-repaired, A-tailed and ligated to custom-designed universal adapters using an end-repair mix, klenow fragment, and T4 DNA ligase (all from Enzymatics). After ligation, adapters were removed by SPRI purification using SPRIselect beads (Beckman coulter) and amplified with Q5 Hot Start DNA polymerase (New England Biolabs) while introducing custom dual indexes. Three biological replicates were sequenced on a NextSeq 500 (Illumina) at a depth of at least 2×10⁷ reads each. Reads were mapped and analyzed with a custom bioinformatic pipeline based on STAR^{63 (link)}, SAMTOOLS^{64 (link)}, and the R packages DEGseq^{65 (link)} and DEseq2^{66 (link)}. We used human genome version GRCh38 and gene annotations from the ENSEMBL release 83. GO analyses were performed using version 6.8 of the DAVID web server^{67 (link), 68 (link)}.