GSM8965288: Deprived P5 Cx3cr1-KO barrel cortex [19L_UB]; Mus musculus; RNA-Seq
The brain tissue was thawed in an ice-cold  glass homogenizer containing 1 mL cell-lysis buffer. After manually homogenizing the samples with 3-5 gentle strokes, samples were  homogenized in a motor-driven overhead stirrer. A post-nuclear supernatant was prepared by centrifugation. Post-mitochondrial supernatant was prepared by centrifugation after NP40 and DHPC treatment. GFP-antibod-coated beads were added to each supernatant. After overnight incubation, the polysome-bound beads were washed with the high-salt buffer. RNA clean-up was performed using RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. RNA integrity was assayed using an RNA Pico chip on a Bioanalyzer 2100 (Agilent, Santa Clara, CA), and only samples with RIN>9 were considered for subsequent analysis. Double-stranded cDNA was generated from 1-5 ng of RNA using the Nugen Ovation V2 kit (NuGEN, San Carlos, CA) following the manufacturer's instructions. Libraries for sequencing were prepared using Nextera XT kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. The quality of the libraries was assessed by 2200 TapeStation (Agilent).