Column purification

Total RNA from fibroblasts and cell lines was extracted with TRIzol (Invitrogen), followed by DNase I treatment (Roche) and column purification (Qiagen). RNA was quantified by spectrophotometry (Nanodrop ND-8000, peqlab), and cDNA was synthesized from 1 μg total RNA with Superscript III reverse transcriptase (Invitrogen) and oligo dT16 primers. Quantitative PCR was performed on a CFX384Touch Real-Time PCR System (Bio-Rad). Delta cycle threshold (Ct) relative quantification, PCR efficiency correction, and multiple reference gene normalization (UBC, TBP, HMBS, and PDHA) were calculated with qBase (Bio-Rad). Oligonucleotides for TRIP11 and LBR were designed using the NCBI Primer Blast software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primer specificity was verified by agarose gel electrophoresis, Sanger sequencing of PCR products, and melting curve analysis.

Nuclear proteins from HeLa wt and HNRNPD KO cl10 were purified as described above for nuclear extract preparation, followed by dialysis over night at 4°C in 50 mM Tris–HCl pH 7.5, 50 mM NaCl, 2 mM MgCl₂, 1 mM DTT and 0.1 mg/ml BSA. A DNA plasmid vector was digested with KpnI (5′ overhangs), HindIII (3′ overhangs) or EcoRV (blunt ends) followed by column purification (Qiagen, Germantown, MD, USA). The reactions were carried out in a final volume of 20 μl with 5 μg of nuclear proteins, per condition, and 300 ng of linearized DNA vector for the indicated time points followed by incubation in 10 mM EDTA, 0.25% SDS and 100 μg/ml proteinase K for 10 min at 37°C. DNA products separated on 0.8% agarose were stained with ethidium bromide.

Total RNA from eWAT adipose tissue was isolated by column purification (QIAGEN) and yield was determined by spectrophotometry (NanoDrop Technologies). From each RNA sample, 200 ng was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). All qPCR data collections were performed using an ABI PRISM 7900 (Applied Biosystems) platform. Relative expression for each target gene was calculated using a standard curve, and all target mRNA gene expression data were normalized to the reference gene, cyclophilin a. Mouse primers were purchased from Integrated DNA Technologies (Coralville, Iowa). Gene and primer sequences are as follows:
m cyclophilin-a fw: 5′-CCACTGTCGCTTTTCGCCGC-3′
re: 5′-TGCAAACAGCTCGAAGGAGACGC-3′
m f4/80 fw: 5′-ATAGCTTCCGAGAGTGTTGTG-3′
re: 5′-TCCAACTGCTCTAACTCTGTG-3′
m cd11c fw: 5′-CTACCCGAGCCATCAATCAG-3′
re: 5′-GCTCTGCTTTCTACTGAGTTCA-3′
m il-6 fe: 5′-TCCTCTCTGCAAGAGACTTCCATCC-3′
re: 5′-AAGCCTCCGACTTGTGAAGTGGT-3′
m mcp-1 fw: 5′-GCAGAGAGCCAGACGGGAGGA-3′
re: 5′-TGGGGCGTTAACTGCATCTGG-3′
m pai1 fw: 5′-CGGCAGAACCCGACAGAGACA-3′
re: 5′-TCCGAGGTCTGGGATGCTGGT-3′
m adpn fw: 5′-AAAAGGGCTCAGGACGCTACT-3′
re: 5′-TGGGCAGGATTAAGAGGAACA-3′
m cd4 fw: 5′-CGTGATAGCTGTGCTCTGAA-3′
re: 5′-CTTCTCTCCATGTCCAACCTAA-3′
m col6a3 fw: 5′-CCCTTCAGTGGTTGAAAGCG-3′
re: 5′-TGAGTCTGCGAACGATCCTG-3′
m tnf-α fw: 5′-AGACCCTCACACTCAGATCA-3′
re: 5′-TCTTTGAGATCC ATG CCGTTG-3′

Total RNA was extracted from virus containing cell lysates using Trizol LS (Invitrogen, Carlsbad, CA) and purified using a column purification method (Qiagen, Germany) in accordance with manufacturer’s recommendations that were modified to isolate both small and large RNA species. RNA concentration and preliminary quality control was estimated on a NanoDrop spectrophotometer (ThermoFisher Scientific) while RNA-Seq was performed commercially (BGI Americas, San Jose, CA) as detailed in Supplementary Methods. Four biological replicates were analyzed from each of the treatments (Uninfected cells treated with DMSO, Uninfected cells treated with RK-33, Infected cells treated with DMSO, and Infected cells treated with RK-33).
RNA-Seq data is in the process of being uploaded to the data server.