GSM4567682: No Injury GFP positive; Mus musculus; RNA-Seq
For a single-cell transcriptional profile, at time of harvesting animals were perfused with ice cold HPBS till kidneys cleared of blood. Kidney halves were immediately placed in cold HPBS with RNasin Plus RNase Inhibitor (0.4U/µl, purchased from Promega N2615 and cut into small pieces (>25). These pieces were immediately transferred into a 2 ml dounce (D8938 SIGMA) loaded with 1ml of NEZ Lysis Buffer (+ Rnase Inh) on ice. Samples were then dounced on ice with 5 strokes of the looser pestle (A) every 2 minutes for 8 mins (25 strokes total). Samples were then dounced 25 times slowly with the tighter pestle (B) on ice. The dounced sample was then filtered through a 40-µm Falcon Nylon Cell Strainer. The filter was then washed with 8 ml of 1% BSA PBS (diluted from SRE0036 SIGMA in CaCl/MgCl free HPBS + Rnase Inh) and nuclei suspension spun in a precooled (4°C) centrifuge at 650g for 8 min. Supernatant was removed and pellet was re-suspended in 1%BSA PBS (+Rnase Inh) and moved to a low bind Eppendorf tube. Nuclei quality and number were assessed with Tryptan Blue staining and a hemocytometer. An appropriate number of nuclei was then pipetted out and spun down again (700g/8min). Supernatant was removed and pellet was resuspended in 2%BSA (+RNase inh) by excessive pipetting to avoid clumping and kept on ice until FACS sorting. Nuclei were sorted on a BD Aria 1 into same 2% BSA solution. Nuclei were first sorted as DAPI positive and then for GFP expression. 10X Genomics Chromium v2 Single Cell 3' protocol:  4,000 singlet nuclei loaded/lane, nuclei capture, lysis and mRNA reverse transcription occurred in-droplets.  cDNA recovered from the emulsion was cleaned-up, amplified by PCR, checked for size and yield on a 4200 Tape station (Agilent), processed into barcoded Illumina ready sequencing libraries and again assayed for size and yield. 10X Genomics Chromium v2 Single Cell 3' protocol
GEO Accession
GSM4567682