title: GSM3473975: P9_wt_3; Mus musculus; RNA-Seq
library_construction_protocol: Brains were enzymatically digested in combination with gentleMACS Octo Dissociator (Miltenyi Biotech),  using Neural Tissue Dissociation kits (P) (130-092-628; Miltenyi Biotech) for embryos at E15.5 and pups at P5,  and Adult Brain Dissociation kits (130-107-677; Miltenyi Biotech) for pups at P9 and adults. After dissociation,  myelin cell debris and erythrocytes were removed according to the manufacturer protocol. ECs were enriched by  depletion of CD45-positive cells with CD45 MicroBeads (30-052-301; Miltenyi Biotech), followed by positive  selection using CD31 MicroBeads (30-097-418; Miltenyi Biotech). The final cell pellets were washed with PBS  and processed for total RNA extraction. Total RNA was isolated using Maxwell RSC simplyRNA cells and  tissue kits (AS1390, Promega), according to the manufacturer protocol. For each timepoint, the RNA-Seq was performed on three indipendent biological replicates.  Libraries for RNA sequencing were prepared following the manufacturer protocols for transcriptome sequencing  with an Ion Proton sequencer (ThermoFisher Scientific). Briefly, 1 μg total RNA was poly-A selected using  Dynabeads mRNA Direct Micro Purification kits (61021; ThermoFisher Scientific), according to manufacturer  protocol. About 50 ng poly-A RNA were used to prepare strand-specific barcoded RNA libraries, with Ion Total  RNA-Seq kits v2.0 (4475936; ThermoFisher Scientific). Briefly poly-A RNAs were fragmented with RNAse III  and purified with Nucleic Acid Binding Beads. After purification, the fragmented poly-A RNAs were hybridized  and ligated with Ion Adaptor, and subsequently reverse transcribed for cDNA preparation. The cDNAs were  amplified with Ion Torrent barcode primer and purified with Nucleic Acid Binding Beads. The final libraries  were quantified on a fluorimeter (Qubit) with HS DNA (ThermoFisher Scientific), and checked for size on an  Agilent Bioanalyzer with HS DNA kits (Agilent).  Four barcoded libraries were pooled together on an equimolar basis to the final concentration of 11 pM,  and clonally amplified using Ion Proton Hi-QTemplate kits (ThermoFisher Scientific). With an IonOneTouch 2  instrument (ThermoFisher Scientific). After emulsion PCR, DNA positive Ion Sphere Particles (ISPs) were  recovered and enriched according to standard protocols, using an IonOneTouch ES instrument (ThermoFisher  Scientific). A sequencing primer was annealed to DNA-positive ISPs and the sequencing polymerase bound,  prior to loading of ISPs into Ion P1 sequencing chips. Sequencing of the samples was carried out according to  the Ion Proton Hi-Q Sequencing kit protocol on and Ion Proton instrument.
attributes.tag: GEO Accession
attributes.value: GSM3473975