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Status |
Public on Jun 08, 2016 |
Title |
KO rep 3 |
Sample type |
SRA |
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Source name |
cerebellum_KO
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Organism |
Mus musculus |
Characteristics |
age: 7-8 weeks background strain: C57BL/6 genotype: alphaT-catenin KO tissue: cerebellum
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Extracted molecule |
total RNA |
Extraction protocol |
After dissection of the brain, cerebellar tissue (n=3) was used for RNA extraction using TRIzol/chloroform during homogenation of the fatty brain tissue. Subsequently, the Aurum Total RNA mini Kit (Bio-rad) was used according to the manufacturer’s protocol. Library construction protocol not provided.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
replicate 3 KO_R3
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Data processing |
The raw data of FASTQ format were processed to remove as much technical artifacts as possible. Low quality ends (phred score <20) were trimmed using the FASTX tool kit (http://hannonlab.cshl.edu/fastx_toolkit). Adapter trimming was performed with cutadapt 1.2.1. Reads shorter than 15 bp after adapter trimming were removed. Quality filtering was done using FastX 0.0.13 and ShortRead 1.16.3. PolyA-reads (more than 90% of the bases equal A), ambiguous reads (containing N), low quality reads (more than 50% of the bases < Q25) and artifact reads (all but 3 bases in the read equal one base type) were removed. The remaining clean reads were aligned against the reference genome of Mus musculus (mus_musculus_GRCm38.73), using the TopHat v2.0.8b software. As parameter options we used: --library-type fr-unstranded –min-intron-length 50 --max-intron-length 500000 –no-coverage-search --no-mixed --read-realign-edit-dist 3. Quality filtering (removal of reads that are non-primary mappings or have a mapping quality < 20), sorting and indexing of the resulting bam-files were done with samtools 0.1.19. Gene expressions of the transcripts were calculated by counting the number of reads in the alignments that overlap with the gene features, using htseq-count 0.5.4p3. As parameters we took: -m union --stranded=reverse -a 0 -t exon -i gene id. Subsequently, the edgeR 3.12.0 tool was utilized to detect the differentially expressed genes between the alphaT-cat WT and KO samples. The modeling of the variance for each gene was done using both the common dispersion model (the same dispersion value is used for each gene) and the moderated tagwise dispersion model (a distinct, individual dispersion is estimated and used for each gene). EdgeR used with the moderated tagwise dispersion model tends to rank more highly as DE genes that are more consistent in their counts within groups. Finally, genes with a p-value ≤ 0.01, corresponding to a false discovery rate (FDR) value ≤ 0.3, were considered as differentially expressed (DE). Genome_build: mus_musculus_GRCm38.73 Supplementary_files_format_and_content: tab-delimited text file including raw counts (output HTSeqCount) for each sample. Excel file contains statistical analysis.
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Submission date |
Jun 07, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Cara J Gottardi |
E-mail(s) |
c-gottardi@northwestern.edu
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Phone |
(312) 503-4123
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Organization name |
Northwestern University
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Department |
Departments of Medicine
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Lab |
Cellular and Molecular Biology
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Street address |
240 East Huron St., McGaw Pavilion, M-323
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City |
Chicago |
ZIP/Postal code |
IL 60611 |
Country |
USA |
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Platform ID |
GPL13112 |
Series (1) |
GSE83084 |
alphaT-catenin in restricted brain cell types and its potential connection to autism |
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Relations |
BioSample |
SAMN05213584 |
SRA |
SRX1829022 |
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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