|
| Status |
Public on Jun 02, 2017 |
| Title |
7T_60_E |
| Sample type |
SRA |
| |
|
| Source name |
Experiment_60m_Telencephalon
|
| Organism |
Gasterosteus aculeatus |
| Characteristics |
FISH id: 7
Sex: male
developmental stage: adult
timepoint in minutes: 60
sample group: Experiment
tissue: Telencephalon
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Heads were flash frozen in liquid nitrogen and the telencephalon and diencephalon were carefully dissected and placed individually in Eppendorf tubes containing 500 μL of TRIzol Reagent (Life Technologies). Total RNA was isolated immediately using TRIzol Reagent according to the manufacturer’s recommendation and subsequently purified on columns with the RNeasy kit (QIAGEN). RNA was eluted in a total volume of 30 μL in RNase-free water. Samples were treated with DNase (QIAGEN) to remove genomic DNA during the extraction procedure. RNA quantity was assessed using a Nanodrop spectrophotometer (Thermo Scientific), and RNA quality was assessed using the Agilent Bioanalyzer 2100 (RIN 7.5-10). RNA was immediately stored at −80 °C until used in sequencing library preparation.
Poly-A RNA was enriched from 1–2 μg of total RNA by using Dynabeads Oligo(dT)25 (Life Technologies), following the manufacturer’s protocol. Two rounds of poly(A) enrichment were performed with a final elution in 14μL of water. The poly-A–enriched RNA was used to prepare RNAseq libraries, using the NEXTflex Directional RNA-seq Kit (dUTP based) with Illumina compatible adaptors (Bioo Scientific). Manufacturer’s instructions were followed and 13–15 cycles of PCR amplification were performed depending on the starting input of total RNA. Libraries were quantified on a Qubit fluorometer, using the dsDNA High Sensitivity Assay Kit (Life Technologies), and library size was assessed on a Bioanalyzer High Sensitivity DNA chip (Agilent). Libraries were pooled and diluted to a final concentration of 10 nM. Final library pools were quantified using real-time PCR, using the Illumina compatible kit and standards (KAPA) by the W. M. Keck Center for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Center (University of Illinois). Single-end sequencing was performed on an Illumina HiSeq 2500 instrument by the W. M. Keck Center for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Center (University of Illinois). The samples were sequenced on 20 lanes.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Fastq files were generated with the software Casava 1.8.2 (Illumina)
read alignment: Tophat 2.0.8, default parameters
read quantification: Reads inside of exon features were counted in union mode using htseq version 0.6.1.
differential expression: Done in R using the Bioconductor package edgeR.
Genome_build: Gasterosteus aculeatus reference genome (the repeat masked reference genome, Ensembl release 75)
Supplementary_files_format_and_content: gacu_mrsb_counts_raw_03.13.2017.txt: Tab delimited text file of the matrix of raw read counts mapped to the mouse genome (Ensembl annotation of the NCBIM37 version of the mouse genome) as created by the edgeR function readDGE. TopHat2 version 2.0.8 (Kim et al. 2013) was used to align reads. Reads inside of exon features were counted in union mode using htseq version 0.6.1 (Anders et al. 2015).
|
| |
|
| Submission date |
Mar 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Alison Bell |
| E-mail(s) |
alisonmb@illinois.edu
|
| Organization name |
University of Illinois at Urbana Champaign
|
| Department |
Animal Biology
|
| Lab |
Alison Bell Lab
|
| Street address |
Justin Smith Morrill Hall, 505 S Goodwin Ave #393
|
| City |
Urbana |
| State/province |
Illinois |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL21984 |
| Series (2) |
| GSE96673 |
Temporal Dynamics of Neurogenomic Plasticity in Response to Social Interactions in Male Threespined Sticklebacks [RNA-Seq] |
| GSE97383 |
Temporal Dynamics of Neurogenomic Plasticity in Response to Social Interactions in Male Threespined Sticklebacks |
|
| Relations |
| BioSample |
SAMN06605438 |
| SRA |
SRX2643210 |
