GSM5664680: KO1_Riboseq; Mus musculus; OTHER
Cortical tissue was homogenized in lysis buffer (containing cycloheximide) and cleared for 10 minutes at 20,000 x g at 4oC.  Monosomes obtained by RNase I digestion were purified using spin-column chromatography. Briefly, MicroSpin S-400 HR columns (Cytiva) were equilibrated with 3 mL of Polysome buffer by gravity flow, then spun down 4 mins at 600 g. 100 µl of RNase I digested lysate was loaded onto the column and spun 2 mins at 600 g. The flow through was collected and the RNA was extracted using RNA Clean & Concentrator-25 kit (Zymo). The rRNA depletion was performed onto linker ligated RPFs using riboPOOLs (siTOOLs).  RNAseq libraries were constructed using SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara), as recommended by the manufacturer. RiboSeq and RNAseq libraries were sequenced on HiSeq4000 (Illumina) at the Center for Advanced Technology (UCSF). total RNA was extracted from mouse cortex using Trizol extraction and quantity was measured on Qubit fluorometer.  Quality of total RNA was checked on the Agilent Bioanalyzer using the RNA 6000 Pico Kit