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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 07, 2012 |
| Title |
K77.5-WT |
| Sample type |
SRA |
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| Source name |
Nkx2-1-iCre targeted eGFP+ cells
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| Organism |
Mus musculus |
| Characteristics |
strain: Swiss
genotype/variation: WT
developmental stage: E14.5
tissue: telencephalon
method: FACsorted cells
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| Treatment protocol |
FACsorting
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| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol followed by RNeasy micro. RNA seq library is prepared for analysis according to the Illumina TruSeq protocol (www.illumina.com). Briefly, 100 ng of Total RNA is purified using poly-T oligo-attached magnetic beads to end up with poly-A containing mRNA. The Poly-A tailed mRNA is fragmented and cDNA is synthesized using SuperScript II. cDNA fragments is end repaired, purified with AMPure XP beads, A-tailed using Klenow exo-enzyme in the presence of dATP. Illumina provided paired end adapters with index are ligated to the A-tailed cDNA fragments and purified using AMPure XP beads. The resulting adapter-modified cDNA fragments are enriched by PCR using Phusion polymerase as follow: 30 s at 98°C, 15 cycles of (10 s at 98°C, 30 s at 60°C, 30 s at 72°C), 5 min at 72°C. PCR products are purified using AMPure XP beads and eluted in 30 µl of resuspension buffer. One microliter is loaded on an Agilent Technologies 2100 Bioanalyzer using a DNA 1000 assay to determine the library concentration and to check for quality. Cluster generation was performed according to the Illumina TruSeq SR Cluster kit v2 (cBot) Reagents Preparation Guide (www.illumina.com). Briefly, 6 RNA-seq libraries were pooled together to get a stock of 10 nM. One microliter of the 10 nM stock was denaturated with NaOH, diluted to 6.5 pM and hybridized onto the flowcell. The hybridized products are sequentially amplified, linearized and end-blocked according to the Illumina Single Read Multiplex Sequencing user guide. After hybridization of the sequencing primer, sequencing-by-synthesis was performed using the HiSeq 2000 with a 36-cycle protocol. The sequenced fragments were denaturated with NaOH using the HiSeq 2000 and the index-primer was hybridized onto the fragments. The index was sequenced with a 7-cycle protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
alignment: Sequence reads were mapped to the mus musculus reference genome (mm9) via TopHat v1.2.0. Only uniquely mapping reads were taken into account.
counts: HTSeq v0.5.3p3 was used to count the number of genes for annotated genes. HTSeq was used in the “union” mode, with gene annotations from Ensembl release 62, grouping exons together based on gene_name (i.e., MGI symbols for protein-coding genes).
differential expression: normalization of gene expression levels and differential gene expression between WT and KO was calculated with DESeq v1.2.2.
Genome Build:
K77.5-WT-ACAGTG.counts.txt: mm9
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| Submission date |
Feb 07, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Bram Van de Sande |
| Phone |
+32 16 33 01 42
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| URL |
http://med.kuleuven.be/lcb/
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| Organization name |
KU Leuven
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| Department |
Center for Human Genetics
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| Lab |
Laboratory for Computational Biology
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| Street address |
Herestraat 49
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE35616 |
Sip1 in cortical interneuron migration |
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| Relations |
| SRA |
SRX118796 |
| BioSample |
SAMN00783735 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM871703_K77.5-WT-ACAGTG.counts.txt.gz |
131.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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