GSM7664486: free RNA from axons of mouse PCNs expressing tRFP, rep 1; Mus musculus; RNA-Seq
For studying both the transcriptional and the translational levels of the axons and cells, total cytosolic RNA (pool of all the fractions) and polysomal RNA (collection of fractions corresponding to the polysomal peaks) were purified from the polysomal profiles. Both total and polysomal RNA extractions were performed with Direct-zol™ RNA purification Kit (Zymo Research). For free/granule transcriptome and translatome analysis at subcellular resolution, after sucrose gradient fractionation, sucrose fractions not associated to polysomes containing RNPs, ribosomal subunits and 80S were pooled in the “free/granule fraction”. Sucrose fractions containing polysomes were pooled into the “polysome fraction”. Trizol was used to extract the RNA. After purification, the RNA was resuspended in DNAse-RNAse nuclease free-water and quantified at the Agilent 2100 Bionalyzer by using the Agilent RNA 6000 Pico kit. The libraries were obtained using Ovation® Single Cell RNA-Seq System (Nugen) or Ovation® SoLo® RNA-Seq library preparation kit (Tecan) according to the manufacturer's protocol. The qualitative and quantitative control of the libraries was performed with High Sensitivity DNA Chip (Agilent Technologies) before sequencing. RNA sequencing was performed with Illumina HiSeq 2500.