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Sample GSM3473969 Query DataSets for GSM3473969
Status Public on Feb 12, 2019
Title P5_wt_3
Sample type SRA
Source name freshly isolated brain endothelial cells
Organism Mus musculus
Characteristics developmental stage: Pups, P5
cell type: Freshly isolated brain endothelial cells
strain: Sox17flox/flox/Cdh5(PAC)-Cre-ERT2- (wt)
Growth protocol n/a
Extracted molecule polyA RNA
Extraction 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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Ion Torrent PGM
Description Pups were treated subcutaneously with 100 μg tamoxifen in the neck skin for every pup of the litter on postnatal day 1 (P1);
Data processing Iontorrent BaseCaller was used for basecalling
samtools (v1.5) was used for extracting sequencing data in fastq format from bam files.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using Trim Galore (v0.4.1) with parameters trim_galore --fastqc --clip_R1 5 --three_prime_clip_R1 5
Trimmed reads were mapped to mm10 whole genome using STAR v 2.5.3a
transcripts per million (TPM) were calculated based on simple formula from Pachter L, 2011.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample
Submission date Nov 15, 2018
Last update date Feb 12, 2019
Contact name Elisabetta Dejana
Organization name Uppsala University
Department Immunology, Genetics and Pathology
Street address Dag Hammarskjolds väg 20
City Uppsala
ZIP/Postal code 751 85
Country Sweden
Platform ID GPL16331
Series (1)
GSE122564 Fine tuning of Sox17 and canonical Wnt coordinates the permeability properties of the blood-brain barrier
BioSample SAMN10430495
SRA SRX5010804

Supplementary file Size Download File type/resource
GSM3473969_EC-P5_WT_3_ReadsPerGene.norm.txt.gz 1.3 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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