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| Status |
Public on Oct 23, 2015 |
| Title |
D5 |
| Sample type |
SRA |
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| Source name |
medial prefrontal cortex
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
tissue: medial prefrontal cortex
gender: Female
estrous cycle: Diestrus
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| Treatment protocol |
Following five days of habituation and handling under pair-housing, the estrous cycle of female rats was monitored by daily vaginal smearing and subsequent cytological analysis for a minimum of two cycles.
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| Growth protocol |
N/A
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| Extracted molecule |
total RNA |
| Extraction protocol |
Brains were dissected out, flash frozen, and stored at -80C until sectioning on a cryostat at -20C for tissue-punching of the medial prefrontal cortex. Total RNA was then extracted using TRI-Reagent according to the manufacturer's protocol (Molecular Research Center, #TR 118), followed by DNAse I treatment to remove any eventual DNA contamination and clean-up (RNA Clean & Concentrator, Zymo Research, Irvine, CA, USA) .
RNA-seq libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina with poly(A) mRNA purification from one microgram of total RNA based on magnetic beads, cDNA synthesis using random hexamers, and final amplification using barcoded primers following the manufacturer’s protocol (#E7530, New England Biolabs, Ipswich, MA, USA). In order to determine the lower limit of detection and verify the linearity of quantification during the statistical analysis of the sequencing data, synthetic RNA Spike-Ins (#4456739, ERCC ExFold RNA Spike-In Mixes, Life technologies, Carlsbad, CA) were added to each sample prior to poly(A) mRNA purification following the recommended dilutions (2 µL of a 1:100 dilution). Of note, Mix 1 and Mix 2 of the ERCC ExFold RNA Spike-Ins were equally distributed among samples in an exclusive manner. The resulting barcoded and unstranded libraries were quantified using a KAPA qPCR library quantification kit (KAPA Biosystems, Boston, MA, USA) with three serial dilutions ran in duplicates on a CFX96 real-time PCR instrument (Bio-Rad). Finally, the absence of adapters or primers contamination was verified on a Bioanalyzer using a DNA High Sensitivity chip (Agilent Technologies, Santa Clara, CA, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
DESeq2_normalized_counts.txt
edgeR_normalized_counts.txt
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| Data processing |
Illumina Casava1.9 software used for basecalling.
Trimmomatic v0.32 was used to remove adapter sequences, and quality filtering using the following parameters: LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:36. Reads shorter than 36 bp after trimming were thus removed from the study.
Filtered reads were then aligned to the reference genome using tophat v2.0.11 and the --b2-very-sensitive Bowtie2 (v2.1.0) preset, guided by GTF file. Of note, reads were aligned to the reference genome to which theERCC spike-ins were added.
The number of reads mapping uniquely to each gene was counted by HTSeq-count v0.6.1p1, and processed for statistical analysis using the R Bioconductor packages edgeR (v3.6.7), and DESeq2 (v1.4.5).
Genome_build: Rnor_5.0 Ensembl Release 76 (August 2014)
Supplementary_files_format_and_content: DESeq2_normalized_counts.txt: Tab-delimited text file contain normalized read counts for each sample, as computed by DESeq2.
Supplementary_files_format_and_content: edgeR_normalized_counts.txt: Tab-delimited text file contain normalized read counts for each sample, as computed by edgeR.
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| Submission date |
Jun 11, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Mohamed Kabbaj |
| E-mail(s) |
mohamed.kabbaj@med.fsu.edu
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| Phone |
850-644-4930
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| Organization name |
Florida State University
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| Department |
Biomedical Sciences
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| Street address |
1115 W Call St
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| City |
Tallahassee |
| State/province |
Florida |
| ZIP/Postal code |
32306 |
| Country |
USA |
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| Platform ID |
GPL20084 |
| Series (2) |
| GSE69765 |
The influence of the estrous cycle on sex differences in the rat adult medial prefrontal cortex transcriptome [RNA-Seq] |
| GSE69773 |
The influence of the estrous cycle on sex differences in the rat adult medial prefrontal cortex transcriptome |
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| Relations |
| BioSample |
SAMN03769765 |
| SRA |
SRX1056227 |
| 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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