title: GSM9160066: C5: ureter ligation last for 7 days; Rattus norvegicus; RNA-Seq
library_construction_protocol: kidney tissues were thawed at 4 ◦C and homogenized using a grinding pestle. A 100 μL aliquot of the homogenized sample was transferred into a 2 mL centrifuge tube containing 400 μL of methanol, followed by vortexing for 1 min. The sample was then centrifuged at 10,000 g for 10 min at 4 ◦C. The supernatant was transferred into a new 2 mL centrifuge tube and concentrated until dry. The residue was reconstituted in 150 μL of 2‑chloro-L-phenylalanine (4 ppm) solution prepared in 80 % methanol water (stored at 4 ◦C), filtered through a 0.22 μm membrane, and transferred to a vial for LC-MS analysis. The total RNA was extracted from kidney tissues using RNAiso Plus (TaKaRa, Dalian, China). The RNA quality and integrity were evaluated and high-quality mRNA from samples were used to construct libraries. The mRNA in the sample was enriched using magnetic beads with oligo (dT) and fragmented into short segments. The synthesis of the first strand cDNA utilized a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-), followed by the subsequent performance of second strand cDNA synthesis using DNA Polymerase I and RNase H. After 3′ -end repair, ligation of a poly(A)-tail to the sequencing linker, and selection of the fragment sizes, the double-stranded cDNA was purified and PCR amplified. Then, the PCR products were purified using the AMPureXP system (Beckman Coulter, Beverly, MA, USA) for sequencing.