RNA-Seq of wild-type murine primary cortical neurons co-cultured in a transwell-based system with Mecp2 knock-out or wild-type cortical murine astrocytes
RNA-Seq of wild-type murine primary cortical neurons co-cultured in a transwell-based system with Mecp2 knock-out or wild-type cortical murine astrocytes
Cortical neurons at Div14 were lysed in Purozol (cod.7326890; Bio-rad) on ice and samples stored at -80°C. Cortical WT neurons were seeded on 6-well plates and cultured in neuronal medium under the influence of WT or Mecp2 KO cortical astrocytes, which were seeded on transwell inserts. When neurons were at Div0, transwell inserts with astrocytes were transferred on cultured cells and the co-cultures were maintained until Div14, without medium supplementation at Div7. Cortical WT neurons were cultivated from Div0 to Div14 under the influence of paracrine factors secreted by Mecp2 KO astrocytes, or WT astrocytes. Neurons cultivated alone were also included. RNA was extracted by using Purezol (cod.7326890; Bio-rad) following . DNasi I Amplification Grade (cod.AMPD1; Sigma-Aldrich) was used to remove eventual genomic DNA contamination and a second RNA extraction was performed in Purezol. RNA integrity was verified using the RNA 6000 Nano Kit on a Bioanalyzer (Agilent Technologies); all samples had a RIN>9. RNA was quantified using Qubit RNA BR Assay Kit. RNA-seq libraries were generated using the TruSeq stranded mRNA kit (Illumina) from 400 ng of RNA samples, after poly(A) capture and according to manufacturer's instructions. Quality and size of RNAseq libraries were assessed by capillary electrophoretic analysis with the Agilent 4200 Tape station (Agilent). Libraries were quantified by real-time PCR against a standard curve with the KAPA Library Quantification Kit (KapaBiosystems, Wilmington, MA, USA).