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Sample GSM1249112

Query DataSets for GSM1249112

Status

Public on Nov 18, 2013

Title

S168_GFPplus_1

Sample type

SRA

Source name

Lateral cortex of the brain

Organism

Mus musculus

Characteristics

strain: C57BL/6
developmental stage: Embryonic day 14.5
cell type: Neurons_Tubb3 positive
genotype: Btg2GFP/Tubb3GFP

Extracted molecule

polyA RNA

Extraction protocol

E14.5 Btg2GFP/Tubb3GFP cortices were dissociated using the papain-based neural dissociation kit (Miltenyi Biotec) after removal of meninges and ganglionic eminences. FACS was performed at 4˚C in the 4-way purity mode with a flow rate of 20 μl/min using side and forward scatter light to eliminate debris and aggregates and gating established for green (488 nm) and red (561 nm) fluorescence. About 1x106 sorted cells from >3 embryos from different litters were immediately lysed in μMACS™ mRNA Isolation Kit and lysates cleaned on LysateClear Colums (Miltenyi) resulting in ca. 1 µg of poly-A RNAs with a integrity number >9.2
Library preparation and enrichment was performed using 15 µl Sera-Mag Oligo(dT) beads (Thermo Scientific) in 50 µl 10 mM Tris-HCl. Samples were treated with 1U Turbo DNAse (Ambion) and purified with Agencourt RNAclean XP beads. Eluted mRNA (18 µl) was chemically fragmented with NEBNext-Mg RNA Fragmentation Module (New England Biolabs), re-purified with RNAclean XP beads and eluted in 13,5 µl nuclease-free water. First strand cDNA synthesis was performed using 0.15 µg/µl Random Primers (New England Biolabs), 1x First Strand Synthesis Reaction Buffer (New England Biolabs), 10 U/uL Superscript II (Invitrogen) with an initial hybridization for 5 min at 65°C with mRNA and primers followed by incubation at 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. After purification with Agencourt Ampure XP-beads (Beckman Coulter), second strand synthesis was performed using the Second Strand Synthesis module (New England Biolabs) replacing the 2nd strand synthesis buffer with a NTP-free buffer and adding equimolar 2.5 mM of d-nucleotides. Incubation for 2.5 h at 16°C was followed by Ampure XP beads purification as described above. End-Repair was done with the NEBnext End Repair Module (New England Biolabs) followed by XP beads purification and A-Tailing using the NEBnext dA-Tailing Module. Adaptors were ligated (Adaptor-Oligo 1: 5'-ACA-CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-3', Adaptor-Oligo 2: 5'-P-GAT-CGG-AAG-AGC-ACA-CGT-CTG-AAC-TCC-AGT-CAC-3') using 1x NEBnext Quick Ligation Buffer (New England Biolabs), 0.3 uM DNA Adaptors, 1 uL Quick T4 DNA Ligase (New England Biolabs) in 50 µl. XP beads purification was followed by dUTP cleavage with 1 U USER enzyme mix (New England Biolabs) per sample and direct enrichment using the PCR Enrich Adaptor Ligated cDNA Library module (New England Biolabs) with indexed primers.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Strand-specific RNA-Seq

Data processing

Basecalling: Illumina CASAVA-1.8.0
Read Alignment: BWA VN:0.5.9-r32-MPI-patch2 against mm9 transcriptome (with exon-exon junction library based on Ensembl Gene v. 61 annotation)
Counts per feature: Reads per gene were computed using bedtools version 2.11, based on Ensembl Gene v. 61 annotation (intersectBed -f 0.55 for non-split reads, -f 1 for split reads - falling on exon-exon junctions).
Read count normalization: The raw read counts (absolute number of reads) were normalized with the DESeq R package (v.1.8.1).
Genome_build: mm9
Supplementary_files_format_and_content: tab delimited file with sample size normalized counts per gene (rows) and sample (columns)

Submission date

Oct 23, 2013

Last update date

May 15, 2019

Contact name

Federico Calegari

E-mail(s)

federico.calegari@tu-dresden.de

Organization name

TU Dresden

Department

Center for Molecular and Cellular Bioengineering (CMCB)