GSM9752424: C57BL/6J, Scr, HC, Rep 4; Mus musculus; RNA-Seq
Whole bilateral hippocampi from 10-12 week old male C57BL/6 mice were homogenized in 1xPBS containing 1x Protease Inhibitor Cocktail (1xPIC; Cell Signaling #5871) and crosslinked in 1% formaldehyde for 5min at RT, then quenched with 125mM glycine for 5min at RT and spun at 2000g for 3min at 4ºC. Pellets were washed 3x with 200uL ice-cold PBS containing PIC, then resuspended in 1mL ice-cold LB1 (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) containing PIC and rotated for 10min at 4ºC. Nuclei were collected by centrifugation at 2000g for 5mins at 4ºC, washed by rotating for 5 min at 4ºC in 200uL TM2 (10mM Tris-HCl pH 8.0, 2mM MgCl2) containing PIC, then resuspended in 200uL TM2 and warmed for 5mins at 37ºC before adding 1mM CaCl2. ChIP-seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit following manufacturer's protocol. Input samples were merged before library preparation. Total RNA was extracted from AAV-shRNA infected dorsal hippocampal tissue using Qiagen RNeasy kit. Eluate was sent to CD Genomics (Shirley, NY) for polyA selection and library prep Library preparation performed at CD Genomics (Shirley, NY)