title: GSM6398721: Obese 2; Rattus norvegicus; RNA-Seq
library_construction_protocol: Kidneys were harvested, cut into quarters, snap frozen and stored at -80 °C for further analysis. Nuclei were isolated using lysis buffer containing 50% ST buffer (292 mM NaCl, 20 mM Tris-HCl pH 7.5, 2 mM CaCl2 and 42 mM MgCl2 in ultrapure water), 2% Nonidet P40 Substitute, 0.2% ultrapure BSA (50 mg/mL, AM2616, Thermofisher Scietific) and 1% Protector RNase inhibitor (3335399001, Sigma Aldrich). 10-30 mg of frozen kidney tissue was minced with a razor blade into 1-2 mm pieces in 1 mL of lysis buffer. Then, chopped tissue was transferred to a dounce homogenizer. After adding 1 mL of lysis buffer tissue was homogenized using pestle A and B (10 times each). The homogenized tissue was filtered through a 40 µm strainer (08-771-1, Fisher Scientific) and the strainer was washed with 2 mL wash buffer (containing 50% ST buffer, 0.2% ultrapure BSA 50 mg/mL, and 1% protector RNase inhibitor). The washed content was centrifuged at 500 g for 5 minutes at 4 °C. Next, the pellet was resuspended in wash buffer, filtered through a 40 µm Flowmi cell strainer (BAH136800040-50EA, Sigma Aldrich). Intact nuclei shape was confirmed under a microscope, and nuclei were counted. 10,000 nuclei were loaded into the Chromium Controller (10X Genomics, PN-120223) on a Chromium Next GEM chip G Single Cell Kit (10X Genomics, PN-1000120) to generate single-cell gel beads in the emulsion (GEM) according to the manufacturer's protocol (10X Genomics, PN-1000121). The cDNA and library was made using the Chromium Next GEM Single Cell 3′ GEM Kit v3.1 (10X Genomics, PN-1000121) and Single Index Kit T Set A (10X Genomics, PN-120262) according to the manufacturer's protocol. Quality control for the libraries was performed using Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) for qualitative analysis. Libraries were sequenced on an Illumina Novaseq 6000 system with 2 × 150 paired-end kits using the following read length: 28 bp Read1 for cell barcode and UMI, 8 bp I7 index for sample index and 91 bp Read2 for transcript.