Illumina NovaSeq 6000 sequencing; RNA-seq of mouse cortical tissue and astrocytes after anesthesia and induction of neuronal activity
RNA-seq of mouse cortical tissue and astrocytes after anesthesia and induction of neuronal activity
For anesthesia/awake samples, mice were culled and cortices rapidly dissected for RNA-seq or TRAP-seq. For visual deprivation and light stimulus samples, mice were decapitated and visual cortices rapidly dissected for RNA-seq or TRAP-seq. For primary cortical neuronal cultures, cultures were placed into serum-free base medium only (control samples) or 100nm tetradotoxin (TTX samples) for 24 hrs, then gathered for RNA-seq. RNA extraction was carried out using the High Pure RNA Isolation Kit (Roche) as per manufacturer's instructions. 3 wells of a 24 well cell-culture plate were lysed per sample and transferred into glass- fibre filter column assemblies. Following DNAse incubation for 15 minutes, the columns were washed, dried and RNA eluted in 30 μL of elution buffer. Low-yield RNA extraction (for example following ribosomal pulldown) was carried out using the Absolutely RNA Nanoprep kit (Agilent) as per manufacturer's instructions. Samples were lysed in 100 μL of lysis buffer with β-mercaptoethanol; mixed with equal volume of 80% sulfolane and added to silica-based fibre filter columns. DNAse treatment was carried out for 15 mins; columns were washed and dried, and RNA eluted in 20 μL of pre-warmed 600C RNAse free water. Total RNA sequencing library preparation was performed by Edinburgh Genomics using Trueseq Stranded Total RNA V2 library preparation. At least 1 μg RNA per sample was utilised, with RNA-integrity number (RIN) > 7.  TRAP-sequencing library preparation (along with sequencing of matched inputs where appropriate) was performed by Cambridge Genomic Services, utilising the Clonetech - SMART-Seq v4 Ultra Low input RNA library preparation.At least 1ng RNA was used per sample with RIN > 7.