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| Status |
Public on Mar 19, 2015 |
| Title |
P11_WT_Retina_1 |
| Sample type |
SRA |
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| Source name |
Retina, postnatal day 11, male
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| Organism |
Mus musculus |
| Characteristics |
genotype: wild type
tissue: retina
age: postnatal day 11
gender: male
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| Growth protocol |
Mice were housed under standard conditions in the animal facility and dissected at the same time point in the light-dark cycle for each dissection.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Retinae were dissected from p11 knockout or wild-type littermate controls. Both retinae from each animal were pooled for each sample. RNA was extracted using trizol and Qiagen Rneasy purification with on-column DNase digestion.
Illumina library construction and sequencing was carried out as follows: total RNA was depleted of ribosomal RNA using the Ribozero rRNA removal kit (Epicentre), heat-fragmented to 200-700 bp in length and cloned using Uricil-N-Glycosylase-based strand-specific cloning. cDNA fragments were sequenced using an Illumina HiSeq 2000
Strand-specific, rRNA depleted total RNA, Paired (WT vs. Mef2d KO) or single-end (WT vs. Crx KO) 49 bp sequencing
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Total RNA was depleted of rRNA by hybridization (using RiboZero) and heat fragmented. Strand-specific RNA-Seq libraries were prepared using the dUTP method, and sequenced on an Illlumina Hiseq instrument
Andzelm-Cherry_MEF2D_Retina_Exon_Densities.txt
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| Data processing |
Base calling was performed using standard approaches.
Illumina raw reads in .fastq files were aligned using bwa 0.6.2. Target sequences included (1) mm9 autosomal and sex chromosomes plus (2) a read-length-dependent splice library (~3M sequences, each 50-98bp). The latter comprised all possible minimal intragenic sequences of two or more exons (based on the RefSeq annotation) for which one or more exon-exon junctions could be crossed by reads of the required length. After bwa-indexing the targets with the bwtsw algorithm, reads were aligned with zero trimming, zero gaps, and allowing up to 5 mismatches. The sam/bam files output by bwa were indexed and sorted and served as input for further processing.
Aligned reads were minimally filtered for QC, removing any reads with uncalled bases or with unmapped or nonuniquely mapped sequences.
Employing "MAPtoFeatures" perl scripts, the loci of uniquely mapped reads were compared to the loci of all genic features (exons, introns, UTRs, CDSs, etc., and all junctions between them) of (1) all genes based on the RefSeq annotation and (2) all rRNA genes based on RepeatMasker, for the alignment target genome. Average exon Density (read coverage per bp) was calculated, equal to rdbp per feature length. Each sample's Densities were renormalized from total reads not in noncoding genes and not in rRNA to a standard total of 10M reads, and to a standard read length of 35bp. With these conventions, units of Density always equal RPKM times 0.35. Quantile normalized exon densities were treated as expression levels comparable among all samples. For paired-end samples only "L" files were considered for determining exon densities.
Genome_build: For all samples mm9 (NCBI37, Jul7. 2007).
Supplementary_files_format_and_content: Entrez gene ID, refseq gene name, chromosome, orientation (strand), gene start, gene stop, refseq number and exon densities are listed for each of the genes analyzed across expression platforms in this study.
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| Submission date |
Sep 12, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Timothy J Cherry |
| Phone |
6174705859
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| Organization name |
Seattle Children's Research Institute
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| Department |
Developmental Biology and Regenerative Medicine
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| Lab |
Cherry Lab
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| Street address |
1900 9th Ave.
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98101 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE61389 |
MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers (RNA-Seq) |
| GSE61392 |
MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers |
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| Relations |
| BioSample |
SAMN03067454 |
| SRA |
SRX700307 |
| 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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