GSM7092016: Control3, mRNA-derived cDNA; Mus musculus; RNA-Seq
The kidneys were decapsulated and digested with HBSS, collagenase and DNase. The red blood cells were removed with a red blood cell lysis buffer. Cells were passed through a 40 µM filter. The filters were rinsed with PBS and thr flow through was pelleted. The cells were resuspended in PBS with viability dye (Viobility 405/452 Fixable Dye, Miltenyi Biotec, Bergisch Gladbach, Germany). 20 minutes after incubation in the dark, PBS is added to the mixture and resuspended. The cells were pelleted by centrifugation at 300g for 10 minutes. Cells were resuspended in ice cold fluorescence-activated sorting (FACS) buffer. The single cell suspension was incubated with Fc Block (Miltenyi Biotec, Bergisch Gladbach, Germany) and then incubated with antibodies.  Countbright beads (Molecular Probes; Invitrogen, Waltham, United States) was added. Flow cytometry acquisition was performed on BD-LSR Fortessa cytometer (Becton Dickinson, Franklin Lakes, NJ, United States of America).  The cells were stained with anti-CD45 – PE-Vio770 (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then washed with PBS and sorted using the BD Influx Cell Sorter (Becton Dickinson, Franklin Lakes, NJ, United States of America) (BD Biosciences, Sortware software) (pressure 20 psi, nozzle 100µm). The single cell suspensions were prepared by following the recommendations of the Single Cell 3' Reagent Kit User Guide (10X Genomics, Pleasanton, California, United States of America). The single cell suspension underwent barcoding and cDNA library preparation through 10X Chromium Controller Instrument (10X Genomics, Pleasanton, California, United States of America) according to the manufacturer's instructions (CG000206 protocol). The Next Gel Bead-In-Emulsion (GEMs) technology (10x Genomics, Pleasanton, California, United States of America) provides a reagent delivery system where cells are individualized into nanoliter scale droplets with one bead, after which preparation of sequencing libraries is performed such that all the cDNA generated from one cell share a common 10X barcode. GEMs were reverse transcribed in a C1000 Touch Thermal Cycler (BioRad) programed at 53°C for 45 min, 85°C for 5 min, and held at 4°C. Unique Molecular Identifiers (UMIs) are added at the same step to allow the discrimination of the PCR duplicates. After reverse transcription, single-cell droplets were broken, and cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNA was then amplified with a C1000 Touch Thermal Cycler programed at 98°C for 3 min, 12 cycles of (98°C for 15 s, 63°C for 20 s, 72°C for 1 min), 72°C for 1 min, and held at 4°C. Subsequently, to construct the Gene Expression library, the amplified cDNA was fragmented, end-repaired, A-tailed, index adaptor ligated, and cleaned with cleanup mix containing SPRIselect Reagent Kit (Beckman Coulter) in between steps. Post-ligation product was amplified and indexed with a C1000 Touch Thermal Cycler programed at 98°C for 45 s, 15 cycles of (98°C for 20 s, 54°C for 30 s, 72°C for 20 s), 72°C for 1 min, and held at 4°C. Besides, the amplified cDNA derived from the barcodes attached to the antibodies is used to construct the Cell Surface Protein library, after addition by PCR of the indexed adapters, with a C1000 Touch Thermal Cycler programed at 98°C for 45 s, 15 cycles of (98°C for 20 s, 54°C for 30 s, 72°C for 20 s), 72°C for 1 min, and held at 4°C.  All the libraries were finally cleaned up with SPRIselect beads and controlled on a Fragment Analyzer HS-NGS run (Agilent). 17     All 10x libraries were pooled and charged with 1% PhiX on one SP lane of the NovaSeq 6000 instrument (Illumina) using the NovaSeq 6000 SP Reagent Kit v1.5 (200 cycles), and the following sequencing parameters: 28 bp read 1 – 8 bp index 1 (i7) – 150 bp read 2. The SP lane generated the following mean reads per cell: 3766, 3301, 3299 and 1865, 2585 and 4547 cells for 3 Control and 3 RM-AKI mice, respectively. OTHER: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)