Wt1os lncRNA as a Novel Regulator of Podocyte Function (CUT&TAG of mouse glomeruli)
Wt1os lncRNA as a Novel Regulator of Podocyte Function (CUT&TAG of mouse glomeruli)
Nuclei were isolated from freshly isolated glomeruli using Nuclei EZ Prep buffer (Sigma-Aldrich) supplemented with protease inhibitors. Isolated nuclei were bound to 10 µl activated Concanavalin A beads (Polysciences, Warrington, USA) per sample for subsequent CUT&Tag processing. Nuclei were incubated overnight at 4 °C with anti-H3K27ac antibody or Normal Rabbit IgG control at 1:50 dilution in Wash150A buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 2 mM EDTA, protease inhibitors). A secondary anti-rabbit IgG antibody was added for 30 min at room temperature at 1:100 in Wash150 buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, 5 mM sodium butyrate, protease inhibitors). pAG-Tn5 (1:20) in Wash300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, 5 mM sodium butyrate, protease inhibitors) was incubated with nuclei for 1 h at room temperature. Tagmentation was carried out for 1 h at 37 °C in Wash300 buffer. Reactions were stopped with 10% SDS, 0.5 M EDTA, and 20 mg/ml Proteinase K, followed by incubation at 1 h at 55 °C. DNA was purified using ChIP DNA Clean & Concentrator (Zymo). To determine library PCR cycle number, a parallel CUT&Tag reaction was tested with 5 µl DNA, 1 µl Universal i5 primer (10 µM), 1 µl Barcoded i7 primer (10 µM), 1.25 µl SYBR Green, 12.5 µl NEBNext HiFi PCR mastermix (NEB, USA), and 4.25 µl nuclease-free water. Libraries were amplified on a QuantStudio 5 system (Applied Biosystems) with 5 min at 72 °C, 30 s at 98 °C, then 39 cycles of 98 °C for 10 s and 63 °C for 10 s, followed by 72 °C for 1 min. 