GSM5823260: 9605; Mus musculus; RNA-Seq
After p erfusion 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. Cells were centrifuged at 1300rpm (330 X g) for 5 minutes and red blood cells lysed using ACK red blood cell lysis buffer (Quality Biological; 10128-802) at 37ºC for 5 minutes. Cells were spun at 1300 rpm (330 X g), resuspended in 1ml of 1% BSA containing Fc blocking solution (dilution 1:200) and incubated for 30 minutes on ice. After blocking, cells were spun and incubated with the following antibodies in 1% BSA for 30 minutes: BV786 rat anti-mouse CD45 (Catalog #: 557659, 30-F11, BD Pharmigen), eFluor®450 rat anti-mouse F4/80 (Catalog #:48-4801, BM8, eBioscience), APC rat anti-mouse CD11b (Catalog #:17-0112, M1/70, eBioscience), APC/Cy7 rat anti-mouse Gr-1 (Catalog #:557661, RB6-8C5, BD Pharmingen), and Fixable Aqua Dead Cell Stain (catalog no. L34957; Invitrogen). After staining for 30 minutes, cells were spun, washed with 1% BSA, and sorted using a Becton–Dickenson FACSAriaII. We sorted ~25,000 live CD11b+ and F4/80+ cells from individual animals (n=2 per group) into individual BSA coated tubes. Cells were counted and approximately 5,000 cells from each animal were subjected to 10X genomics. This was done for n=2 animals from each experimental group; all animals were female. 10xChromium single cell libraries were prepared according to the standard protocol outlined in the manual. Briefly, sorted single cell suspension, 10x barcoded gel beads, and oil were loaded into Chromium™ Single Cell A Chip to capture single cells in nanoliter-scale oil droplets by Chromium™ Controller and to generate Gel Bead-In-EMulsions (GEMs). Full length cDNA libraries were prepared by incubation of GEMs in a thermocycler machine. GEMs containing cDNAs were broken and all single cell cDNA libraries were pooled together, cleaned using DynaBeads MyOne™ Silane beads (Fisher PN 37002D), and pre-amplified by PCR to generate sufficient mass for sequencing library construction. Sequencing libraries were constructed by following the steps: cDNA fragmentation, end repair & A-tailing, size selection by SPRIselect beads (Beckman Coulter, PN B23318), adaptor ligation, sample index PCR amplification, and a repeat of SPRIselect beads size selection. The final constructed single cell libraries were sequenced by Illumina Nextseq machine with total reads per cell targeted for a minimum of 50,000.