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| Status |
Public on Jun 01, 2014 |
| Title |
AG rep1 |
| Sample type |
SRA |
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| Source name |
lens
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| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6<har>
tissue: lens
age: 15.5 days post conception
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| Extracted molecule |
total RNA |
| Extraction protocol |
Lenses were removed, flash frozen on dry ice, and total RNA was extracted from Sip1 cKO and wild type lenses at E15.5 (30 lenses per biological replicate; three independent biological replicates analyzed for each genotype) using the SV Total RNA Isolation System (Invitrogen). Illumina® TruSeqTM RNA Sample Preparation Kit v2 was used to prepare the sequence libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols
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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 |
Basecalls performed using CASAVA version 1.8.2
Sequenced reads were trimmed for adaptor sequence, and all short (less than 35 base pairs), low-quality, and non-speccific sequences were discarded. Sequences were then mapped to the Mus musculus reference genome (Build NCBI-M37.65 ENSEMBL/MGI annotations) using the CLC RNA-Seq reference mapping algorithm (length parameter: 0.9, identity: 0.8)
Reads per million per kilobase (RPMK) values were calculated to rank gene expression. Observed counts were quantile normalized according to Bolstad et al., Bioinformatics, 2003 and then underwent differential expression analysis using the beta binomial method of Baggerly et al., Bioinformatics, 2003 with False Discovery Rate correction for multiple comparisons according to Benjamini and Hochberg, Journal of the Royal Statistical Society, 1995.
Only genes with mean unnormalized RPMK values greater than two for either the wild type or Sip1 cKO were investigated, estimated to correspond to approximately one mRNA molecule per cell (based on the estimate that typical mammalian cells contain 500,000 molecules of mRNA (Bryant and Manning, 1998) although cells can vary significantly in mRNA content (Islam et al., 2011)). A minimum change in RPMK between the wild type and Sip1 conditional knockout greater than two was also used in further analysis. Additionally, only genes with a p-value less than 0.05 (for a 95% confidence interval) and more than a 2.5 fold change in normalized RPMK value are reported. This fold change threshold was chosen based on experimental data comparing relative gene expression between E15.5 lenses from mixed background Sip1 f/f no Cre control mice and inbred C57Bl/6<har> mice (also submitted to GEO) which indicated that a large proportion of changes below 2.5 fold are likely due to variation in genetic background, not the Sip1 gene deletion.
Genome_build: Build NCBI-M37.65 ENSEMBL/MGI annotations
Supplementary_files_format_and_content: tab-delimited text files include normalized and non-normalized RPKM values for each Sample as well as gene annotation (ID, name, synonyms, and MGI identifier)
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| Submission date |
Aug 16, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Melinda K. Duncan |
| E-mail(s) |
duncanm@udel.edu
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| Phone |
3028310533
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| Organization name |
University of Delaware
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| Department |
Biological Sciences
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| Lab |
Vertebrate Development
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| Street address |
327 Wolf Hall
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| City |
Newark |
| State/province |
DE |
| ZIP/Postal code |
19716 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE49949 |
Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development |
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| Relations |
| BioSample |
SAMN02318583 |
| SRA |
SRX336064 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1210567_AG1.txt.gz |
276.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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