signature=118e844de9fea3f1fc98782813f19425,Development of a new macrophage-specific TRAP mouse (Mac

Generation of transgenic c-fms-eGFP-L10a mice and maintenance

We first engineered an eGFP-L10a transgene under the control of the macrophage-specific promoter c-fms using standard cloning techniques. An adapter was added to the c-fms promoter (Topo cloning) to create a new Pac1 restriction site. The eGFP-L10a- cassette was integrated into the backbone of the c-fms plasmid following prior Pac1/Sal1 digestion. The construct (Sup. Fig. S1) was then sequenced and validated in vitro. Finally, Mlu1/Sal1 digestion produced the linearized complete transgene containing the c-fms promoter followed by the eGFP-L10a encoding sequence which was then successfully integrated into the genome of F1-hybrid mouse embryos (Gene Modification Facility, Harvard University) via pronuclear injection. All mouse studies were performed according to the animal experimental guidelines for laboratory mice issued by the Animal Care and Use Committee (IACUC) at Harvard University and Philipps-University Marburg. The experimental protocol was approved by the IACUC committee at Harvard University. Transgenic mice were maintained on a mixed C57BL/6JxDBA/2 background. Genomic DNA was obtained from tail biopsies and genomic integration of the c-fms-eGFP-L10a transgene verified via PCR-based genotyping using the following primers: sense: 5′-GGCATCGACTTCAAGGAGGA-3′, antisense: 3′-GGTCGTAGTTCTTCAGGCTGA-5′.

Unilateral ureteral obstruction (UUO)

UUO surgery was performed as previously described

Mouse tissue preparation and histology

Mice were euthanized under deep isoflurane anesthesia and immediately perfused via the left ventricle with ice-cold PBS for 1 min. Kidneys were harvested, cut in transversal sections, fixed in freshly prepared 4% paraformaldehyde and 5 μm cryosections (Tissue Tek, Sakura) were stained using the following primary antibodies: rat anti-F4/80 (Abcam, 1:1000), rat anti-CD31 (eBioscience, 1:600), rabbit anti-CD3 (eBioscience, 1:300), rabbit anti-laminin (Sigma, 1:2000), rat anti-PDGFRβ (eBioscience, 1:800), mouse anti-αSMA (Sigma, 1:2500), rat anti-Ly6g (BD Pharmingen, 1:500). Sections were subsequently incubated with corresponding Cy3- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch, 1:400). For PAS staining, kidney sections were fixed in formalin. Images were captured using Nikon C1 confocal and Zeiss Axio Observer Z1 microscope, respectively.

Western Blot

Western blotting was performed as previously describedTM ECL substrate (Bio-Rad) on the Fusion FX (Vilber) chemiluminescent imaging platform.

RT-qPCR

RNA was extracted from tissue using standard techniques (RNAeasy kit; Qiagen). Purity was determined based on A260/280 ratios (Nano Drop, Thermo Fisher). 1 µg of RNA was reverse transcribed for each sample using iScriptTM reverse transcriptase (BioRad). Real-time quantitative PCR was performed on the ABI Prism 7500 Real-Time PCR System using iTaq universal SYBR® Green reaction mix (Bio-Rad). The specific primers used for RT-qPCR are listed in Sup. Table 10. Expression values were normalized to the housekeeping gene GAPDH.

Translational ribosome affinity purification (TRAP)

Polysomal RNA from whole organ lysates was extracted and purified as previously described

RNA quality control and RNA-Seq

For assessment of RNA quality and yield, purified RNA was measured using the Agilent 2100 Bioanalyzer® system (Agilent Technologies) (Sup. Fig. S6). RNA samples with sufficient yield and RIN ≥ 9 were processed according to an ultra-low input protocol at DKFZ Heidelberg genomics core facility using Illumina HiSeq2000 RNA-Sequencing. Fast QC reports were analyzed after each run for quality control. FASTQ Mean Sequence Quality (Phred Score) was over 36 for each sample.

Computational analysis of RNA-Seq data

The RNA-Seq data were mapped against the mouse reference genome GRCm38 using Hisat2ftp://ftp.ensembl.org/pub/release-89/gff3/mus_musculus/). The resulting files were analyzed with DESeq2

Statistics

Data are shown as mean ± SEM if not otherwise indicated. Statistical analysis was performed and graphs prepared using Prism software. The unpaired Student’s t test was used to determine differences between groups. P values of less than 0.05 were considered statistically significant.