title: GSM5225273: 18min IRI 28 days_1, GFP positive, snRNA-Seq; Mus musculus; RNA-Seq
library_construction_protocol: For a single-cell transcriptional profile, at time of harvesting animals were perfused with ice cold DPBS until kidneys cleared of blood. Kidney tissue was flash frozen in liquid nitrogen for later nuclear isolation. Nuclear isolation was achieved by thawing tissue and cuting into pieces smaller than a grain of rice. These pieces were immediately transferred into a 2 ml dounce (D8938 SIGMA) loaded with 1ml of NEZ Lysis Buffer with RNasin Plus RNase Inhibitor (0.4U/µl,  Promega N2615) and protease inhibitor added, on ice. Samples were then dounced on ice with 15 strokes of the looser pestle and 5 times slowly with the tighter pestle. The dounced sample was then filtered through a 100-µm Falcon Nylon Cell Strainer and spun down in a swinging bucket centrifugre at 500g for 5 min. Supernatant was removed and pellet was re-suspended in NEZ buffer minus protease inhibtor, filtered  through a 40-µm Falcon Nylon Cell Strainer, and  spun down in a swinging bucket centrifuge at 500g for 5 min.  Isolated nuclei were resuspended in a 1% BSA-DPBS +0.4U/ul Rnase Inhibitor supplemented with 1μg/mL Hoechst 33334. Nuclei were then sorted using a BD Aria I with 70μM nozzle. Nuclei were isolated from debris by gating on the Hoechst positive signal, doublets were removed with gating on a FSC-A vs. FSC-H plot and GFP positive gating set by comparison to a negative control. Nuclei were sorted into a BSA-DPBS Rnase inhibitor solution calculated to yield the same input 1% BSA-DPBS solution after dilution by FACS buffer. Flow rate was set at 1-2 and machine was cooled to 4°C throughout the sorts. After sorting nuclei solution was concentrated by spinning down at 300g for 3mins and gently removing buffer from above the pelleting level and resuspending the pellet in the remaining buffer . Post sorted nuclei quality and number was further assessed with Tryptan Blue staining and a hemocytometer and subsequently processed according to either the 10X Chromium SIngle Cell 3' v2 (CG00052) or v3 (CG000183)  user guide protocol . Completed 10X libraries were sequenced using an Illumina NextSeq® 500/550 High Output Kit v2 at The Translational Genomics Center at the Children's Hospital Los Angeles Center for Personalized Medicine. 10X Genomics Chromium Single Cell 3' (v2 or v3) protocol snRNA-Seq