DOI: 10.5061/dryad.51c59zwb7
title: Genome wide mRNA sequencing data in macrophages without and with CX-5461 treatment

description: CX-5461, a novel selective RNA polymerase I inhibitor, shows potential
 anti-inflammatory and immunosuppressive activities. However, the molecular
 mechanisms underlying the inhibitory effects of CX-5461 on
 macrophage-mediated inflammation remain to be clarified. In the present
 study, we attempted to identify the systemic biological processes which
 were modulated by CX-5461 in inflammatory macrophages. Primary peritoneal
 macrophages were isolated from normal Sprague Dawley rats, and primed with
 lipopolysaccharide or interferon-gamma. Genome-wide RNA sequencing was
 performed. The study suggests that limiting cell proliferation
 predominates in the inhibitory effects of CX-5461 on macrophage-mediated
 inflammation. 
description: The RNA processing and
 sequencing services were provided by LC-Bio Technology (Hangzhou, Zhejiang
 Province, China). Total RNA was extracted using Trizol reagent (Thermo
 Fisher) following the manufacturer's instructions. The RNA quantity
 and purity were analyzed using Bioanalyzer 2100 and RNA 6000 Nano LabChip
 Kit (from Agilent, Santa Clara, CA, USA). RNA samples with RIN number
 &gt; 7.0 were used for library construction. Purified mRNA was
 obtained from 5 mg of total RNA using Dynabeads Oligo (dT)
 (Thermo Fisher) with two rounds of purification; the mRNA was then
 fragmented using divalent cations (Magnesium RNA Fragmentation Module from
 New England Biolabs, Ipswich, MA, USA) under 94oC for 5 min. The cleaved
 mRNAs were reverse-transcribed to cDNAs using SuperScript II Reverse
 Transcriptase (Thermo Fisher), which were used as templates to synthesize
 U-labeled second-strand DNAs with<em> E. coli</em> DNA
 polymerase I, RNase H (all from New England Biolabs) and dUTP (Thermo
 Fisher). After ligation with Dual-index adapters, the DNA library was
 treated with heat-labile UDG enzyme (New England Biolabs) to remove the
 U-labeled second strands, and amplified by PCR. The average insert size of
 the final cDNA library was 300 ± 50 bp. Paired-end sequencing (2 × 150 bp)
 was performed using a NovaSeq 6000 Sequencing System (Illumina, San Diego,
 CA, USA). High quality clean reads were obtained by filtering with
 Cutadapt (version 1.9) (https://cutadapt.readthedocs.io/en/stable/) and
 verified with FastQC
 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Read alignment
 to the Rattus norvegicus reference genome was performed using HISAT2
 package (version 2.0.4) (https://daehwankimlab.github.io/hisat2). Read
 assembly was performed using StringTie (version 1.3.4d)
 (http://ccb.jhu.edu/software/stringtie). StringTie and Ballgown
 (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html)
 were used to estimate the relative expression levels (expressed as
 fragment per kilobase per million reads/FPKM values).
description: In each experimental group, 3
 independent samples were included. 
 Sample identity:
 control1-control3: Control
 group IFN1-IFN3: Interferon-gamma-treated group
 IFN_CX1- IFN_CX3: Interferon-gamma + CX-5461
 co-treated group LPS1-LPS3: Lipopolysaccharide-treated
 group LPS_CX1- LPS_CX3: Lipopolysaccharide + CX-5461
 co-treated group Concentrations used:
 CX-5461 (1 uM) IFN-gamma (150 U/mL)
 LPS (1 ug/mL)