Wt1os lncRNA as a Novel Regulator of Podocyte Function (RNA-Seq of mouse glomeruli)
Wt1os lncRNA as a Novel Regulator of Podocyte Function (RNA-Seq of mouse glomeruli)
Mice were sacrificed by cervical dislocation. Both kidneys were collected with the abdominal aorta attached. Kidneys were perfused through the renal arteries with magnetic beads (Dynabeads M-450 Tosylactivated, Thermo Fisher Scientific) in HBSS. Perfused kidneys were minced and incubated in digesting solution (1 mg/mL collagenase type II, 0.75 mg/mL pronase E, and 100 U/mL DNase I in HBSS) for 15 minutes at 37°C. Digested tissue was filtered through a 100-μm strainer twice. Glomeruli were isolated using magnetic separation, washed twice in HBSS, pelleted, and stored at -80°C.  Total RNA was isolated from pelleted glomeruli using the Direct-zol RNA Kit (Zymo Research, USA) according to the manufacturer's protocol. RNA concentrations were measured with the Qubit™ RNA Broad Range assay kit (Invitrogen), and RNA integrity was validated using the Tape Station system (Agilent). Sequencing libraries were prepared with the Illumina Stranded TruSeq Ribo Zero protocol