GSM3825590: R2b; Mus musculus; RNA-Seq
After perfusion of the mouse with PBS, kidneys were minced and digested in 1 ml of RPMI 1640 containing 1 mg/ml collagenase type I (Sigma-Aldrich) and 100 U/ml DNase I (Sigma-Aldrich) for 30 min at 37ºC with agitation. Kidney fragments were passed through a 70-µm mesh (Falcon; BD Biosciences) yielding single-cell suspensions. Red blood cells were lysed and cells were resuspended in 1 ml PBS containing 1% BSA with Fc blocking solution for 30 minutes on ice. 2 X 106 cells were stained for 30 minutes at room temperature with primary antibody. Following staining, R2a and R2b resident macrophages were sorted and collected using a BD FACS AriaII. RNA was isolated from FACS sorted cells using the RNeasy Plus Mini Kit (Qiagen cat. 74134) according to standard procedures. For RNA sequencing, the NEBNext mRNA library prep kit for Illumina with ribosome reduction was used to generate the library, which was subsequently sequenced using Illumina NextSeq500 with paired end 75bp sequencing and aligned to the University of California, Santa Cruz (UCSC) GRCm38/mm10 reference genome using the STAR software package. For each sample, at least 25 million reads were obtained.  Following alignment, HTSeq-count (version 0.9.1) was used to count the number of reads mapping to each gene.  Fragments per kilobase of transcript per million mapped reads (FPKM) were calculated using the Cufflinks suite (https://github.com/cole-trapnell-lab/cufflinks) and differential expression was then applied to the count files using DESeq2.