Deoxyribonuclease

SUM159 NTC, HIF-DKD, and PADI4-KD subclones were seeded into six-well plates in three biological replicates and exposed to 20 or 1% O₂ for 24 hours. Total RNA was isolated from the subclones using TRIzol (Invitrogen) and treated with deoxyribonuclease (Qiagen). Library preparation and sequencing using the NovaSeq 6000 platform (Illumina) were performed by Genetic Resources Core Facility High Throughput Sequencing Center at the Johns Hopkins University School of Medicine (https://grcf.jhmi.edu/). The FASTQ files were subjected to quality check and analyzed by Genialis Inc. (https://www.genialis.com/). Differential expression results with a false discovery rate of <0.05 and mRNA fold change of >1.5 were used as a cutoff for further downstream analysis.

Total ribonucleic acid (RNA) was extracted with the PicoPure RNA isolation kit (Applied Biosystems, Inc., Alameda, California, USA) from 2,000 sorted cells and treated with deoxyribonuclease (Qiagen Inc., Valencia, California, USA). Complement deoxyribonucleic acid was prepared by reverse transcriptase (Invitrogen, Carlsbad, California, USA) with an oligo(dT) primer (Invitrogen). The messenger RNA (mRNA) levels were measured in a total volume of 20 μL by SYBR Green‐based reverse transcription polymerase chain reaction using the StepOnePlus™ Real‐Time PCR System (Applied Biosystems Inc., Foster City, California, USA). Polymerase chain reaction analysis was carried out with 0.2 μmol/L of the primers listed in Table S1. Thermal cycling conditions were denatured at 95°C for 10 min followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. Melting curve dynamics and the absence of primer dimers were confirmed. Peptidylprolyl isomerase A was used as the internal control for normalization. All data are shown using the Δcycle threshold (ΔCT) method (CT [internal control] − CT [target gene]).