GSM2091174: Brain_E135_EC_Translatome_2; Mus musculus; RNA-Seq - SRA

SRX1638467: GSM2091174: Brain_E135_EC_Translatome_2; Mus musculus; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 30.5M spots, 1.6G bases, 1.3Gb downloads

Submitted by: NCBI (GEO)

Study: Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels

PRJNA315441 • SRP071876 • All experiments • All runs

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Purpose: Exploring mechanisms defining the unique nature of vascular development and differentiation in the brain . Methods: High-resolution gene expression profiles of embryonic endothelial cells (EC) using translating ribosome affinity purification (TRAP) and single cell RNA-sequencing. RNA-sequenzing of transcription factor infected HUVEC. Results: Comparisons of different organ-, temporal- or Ctnnb1 knock out-specific vascular translatomes revealed extensive molecular changes during the unique development of the brain endothelium. We identified brain endothelial-specific transcription factors – Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551 and Zic3 – that are associated with the temporal maturation of the blood-brain barrier and act downstream of the Wnt/Ctnnb1 signaling pathway. Profiling individual EC revealed a remarkable heterogeneity. Nevertheless, high levels of Foxf2, Foxq1, Ppard and Zic3 were correlated with elevated expression of differentiation markers. This could be recapitulated in vitro, where expression of Foxf2 and Zic3 in HUVEC led to an induction of blood-brain barrier differentiation markers. Conclusions: Our study provides further insights into the temporal and cellular complexity of the developing embryonic brain endothelium. Our data raises many questions and provides a basis for further studies aiming to unravel cellular and molecular mechanisms that underlie vascular development and differentiation in the CNS. We anticipate that additional investigation of the identified transcription factors is likely to be a fruitful approach in understanding and manipulating the development of BBB and to engineer more advanced blood brain barrier in vitro models. Overall design: TRAP-seq profiles for E11.5 till E17.5 wild type (WT) brain EC; E14.5 and E17.5 endothelial Ctnnb1 -/- brain EC; E14.5 head, heart, kidney, limb, liver and lung WT EC in triplicate, using Illumina HiSeq 2000. Single cell RNA-sequencing of E14.5 brain EC. RNA-sequenzing of HUVEC infected with either control, Foxf2, Foxq1, ZIC3 or three transcription factors combined in triplicate.

Sample: Brain_E135_EC_Translatome_2

SAMN04558551 • SRS1344949 • All experiments • All runs

Organism: Mus musculus

Library:

Instrument: Illumina HiSeq 2000

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: SINGLE

Construction protocol: TRAP: Translated and transcribed RNA from brain (forebrain and part of the diencephalon), head (without brain), heart, kidney, limb, liver or lung was purified from one to six mCherryTRAP embryos (age-dependent) as described (Hupe et al.: Nucleic Acids Res, 2014). HUVEC: RNA was purified using RNeasy® Micro Kit (Qiagen) following the manufacturer’s instructions. Single cell: Forebrains of mCherryTRAP E14.5 embryos were dissected in ice-cold PBS. The tissue was diced with a scalpel and subsequently enzymatically dissociated in PBS containing 1 mg/ml collagenase typ II (Worthington) and 1 µl/ml DNase I (Roche) at 37°C for 30 min with gentle sequential trituration. The cell suspension was filtered through a 40 µm cell strainer and 20 ml of DMEM containing 10 % FBS was added. After centrifugation at 800 x g for 5 min, the cell pellet was resuspended in 2 ml PBS containing 2 % FBS. Cells were sorted on a BD FACSAria III Cell Sorting system (BD Bioscience) based on mCherry fluorescence utilizing the 488-nm laser. mCherryTRAP-negative embryos were used to define background fluorescence. Forward and side scatter analysis were used as gates to avoid cell debris and doublets. Single cells were collected in a 96 well plate contain 4 µl of a mild hypotonic lysis buffer [0.1% Triton X-100 (Sigma), 1 U/µl recombinant RNase inhibitor (Promega), 2.5 µM anchored oligo-dT primer (5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, Biomers), and 2.5 mM dNTP mix (Fermentas)] in each well. TRAP and HUVEC: RNA libraries were prepared for sequencing using standard Illumina protocols Single cell: Libraries were generated following Smart-seq2 (Picelli et al.: Nat Methods, 2013) and Tn5 transposase tagmentation (Picelli et al.: Genome Res, 2014) protocols.

Experiment attributes:

GEO Accession: GSM2091174

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 30.5M spots, 1.6G bases, 1.3Gb

Run	# of Spots	# of Bases	Size	Published
SRR3232763	30,485,186	1.6G	1.3Gb	2017-07-12

ID:: 2342321

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