title: GSM5747809: F14-1; Mus musculus; OTHER
library_construction_protocol: Whole cortex of Aldh1l1-L10-GFP+ mice were used; mice underwent rapid decapitation and cortex was dissected and fixed in cyclohexamide to fix the polysomes (mRNA with ribosomal subunits). Cortical tissues were homogenized and then incubated with biotinylated magnetic beads coated with protein L + anti-GFP to immunoprecipitate astroglial-specific polysomes. Polysomes were lysed and mRNA was extracted (see previous work for detailed methods: Simard et al., (2018) Neuropsychopharmacol.) RNA samples were sent to the Yale K.E.C.K facility for sample QC as per Simard et al. 2018: RNA quality was determined using a nanodrop and RNA integrity was determined by running an Agilent Bioanalyzer gel (RINs >8). mRNA was purified from approximately 500ng of total RNA with oligo-dT beads and sheared by incubation at 94C. Following first-strand synthesis with random primers, second strand synthesis was performed with dUTP for generating strand-specific sequencing libraries. The cDNA library was then end-repaired and A-tailed, adapters were ligated and second-strand digestion was performed by Uricil-DNA-Glycosylase. Indexed libraries that met appropriate cut-offs for both were quantified by qRT-PCR using a commercially available kit: Kapa Library Quant Kit (Illumina) (KAPA Biosystems # KK4854-07960298001) and insert size distribution was determined with the LabChip GX or Agilent Bioanalyzer. Only samples with a yield of ≥0.5 ng/ul were used for sequencing.