title: Illumina HiSeq 2500 sequencing; Single cell RNA sequecing analysis of mouse resident and infiltrating immune cells during acute kidney injury and fibrosis
design: Single cell RNA sequecing analysis of mouse resident and infiltrating immune cells during acute kidney injury and fibrosis
library_construction_protocol: Two established models of warm IRI were used to induce kidney injury. In the first model, female C57BL6 underwent unilateral 30 min ischemia (left kidney) by renal pedicle clamping, with immediate contralateral (right) kidney nephrectomy, defined hereafter as regeneration model. Age and sex matched sham operated mice, without ischemia, but contralateral kidney nephrectomy and naïve non-operated mice were used as controls. In the second model, to induce kidney fibrosis, male C57BL6 underwent unilateral 30 min ischemia (left kidney) by renal pedicle clamping, without contralateral (right) kidney nephrectomy defined hereafter as fibrosis model. Sham animals were only subjected to laparotomy, without renal vessel clamping. Age and sex matched animals were used as sham and naïve controls. 100.000 FACS-sorted kidney CD45+ and between 3.000-10.000 kidney CD4+ cells were resuspended in PBS with 0.05% BSA at a concentration of 1000 cells/ul. Viability was accessed by Trypan blue cell counting and exceeded 90%. 2000-10.000 cells were loaded on a 10x Chromium instrument to generate single cell GEMs. Single cell RNA-Seq libraries were prepared using GemCode Single Cell 3'Gel Bead Kit, according to manufacture's instructions (10x Genomics). Amplified cDNA product was cleaned up with the SPRIselect Reagent Kit (0.6X SPRI). Indexed sequencing libraries were constructed using the reagents in the Chromium Single Cell 3' library kit V2 (10x Genomics). The barcode sequencing libraries were quantified using a Qubit 2.0 TM dsDNA HS Assay Kit (Invitrogen). The quality of the libraries was accessed on a 2100 Bioanalyzer using an Agilent High Sensitivity DNA kit (Agilent).