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GEO help: Mouse over screen elements for information. |
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Status |
Public on Nov 18, 2013 |
Title |
S355_GFPplus_3 |
Sample type |
SRA |
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Source name |
Lateral cortex of the brain
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 developmental stage: Embryonic day 14.5 cell type: Neurons_Tubb3 positive genotype: Btg2GFP/Tubb3GFP
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
Strand-specific RNA-Seq
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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)
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Submission date |
Oct 23, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Federico Calegari |
E-mail(s) |
federico.calegari@tu-dresden.de
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Organization name |
TU Dresden
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Department |
Center for Molecular and Cellular Bioengineering (CMCB)
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Lab |
Calegari Lab
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Street address |
Fetscherstr. 105
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City |
Dresden |
ZIP/Postal code |
01307 |
Country |
Germany |
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Platform ID |
GPL13112 |
Series (1) |
GSE51606 |
Transcriptome Sequencing During Mouse Brain Development Identifies Long Non-Coding RNAs Functionally Involved in Neurogenic Commitment |
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Relations |
Reanalyzed by |
GSE80797 |
BioSample |
SAMN02381196 |
SRA |
SRX367090 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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