|
| Status |
Public on Apr 23, 2019 |
| Title |
KO_81 |
| Sample type |
SRA |
| |
|
| Source name |
mPFC
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
genotype: deletion of Fmr1 exon 8
tissue: medial prefrontal cortex (mPFC)
sample preparation: whole lysate
age: 8 weeks
batch: 1
parents: a1
rnaseqlane: a2
rin: 10
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. Subsequently, the quantity of all purified RNA samples was measured on a nanodrop (2.07±0.01 A260/280; 2.11±0.19 A260/230) and the quality and integrity were measured with the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). All RNA integrity numbers were greater than 9 (9.6±0.3).
1µg of total RNA was used for the preparation of the RNAseq library using the Illumina Genome Analyzer IIx TruSeq mRNA Seq Kit supplied by Illumina (Cat number: RS-122-2001). A poly-A-based mRNA enrichment step was carried out and cDNA was synthesized and used for library preparation using the Illumina TruSeqTM RNA sample preparation kit as previously described (Tariq MA et al. 2011), except for the following step: adapter-ligated DNA fragments were size-selected by gel-free size selection using appropriate concentration of SPRI AMPure beads to get an average 200bp peak size in adaptor ligated DNA. The size selected adaptor-ligated DNA fragments were amplified by LM-PCR. Then, Illumina recommended 6bp barcode bases were introduced at one end of the adaptors during PCR amplification step. The amplified PCR products were then purified with SPRI AMPure XP magnetic beads to get the final RNAseq library, which was used for high-throughput RNAseq. All samples were sequenced on the Illumina Genome Analyzer IIx.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
All high-quality short reads were mapped to the rat reference genome rn4 using the STAR Aligner v2.4.0g1 (Dobin A et al. 2013) with 2-pass mapping strategy (--twopassMode Basic).
RNAseq read quality was checked with the FastQC and RNA-seqQC tools.
Uniquely mapped reads with overlapping genes were counted with featureCounts v1.4.4 (Liao Y et al. 2014) parameters (featureCounts -T 10 -p -t exon -g gene_id)
Filtered raw count data was subjected to conditional quantile normalization (CQN) (Hansen KD et al. 2012) to remove systematic bias introduced by GC-content and correct for global distortions
Normalized data were inspected for outlying samples using unsupervised clustering of samples (Pearson’s correlation coefficient and average distance metric) and principal component analysis to identify outliers outside two standard deviations from these grand averages. Based on these metrics, two outliers were removed from these data (WT=2).
Genome_build: mm4
|
| |
|
| Submission date |
Feb 04, 2019 |
| Last update date |
Apr 23, 2019 |
| Contact name |
Michael S Breen |
| Organization name |
Ichan School of Medicine at Mount Sinai
|
| Department |
Depts. of Psychiatry, Genetics and Genomic Sciences
|
| Street address |
1468 Madison Ave
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL10669 |
| Series (1) |
| GSE126057 |
Deletion of the KH1 domain coding sequence of Fmr1 leads to transcriptional alterations and attentional deficits in rats |
|
| Relations |
| BioSample |
SAMN10869267 |
| SRA |
SRX5329774 |
