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| Status |
Public on May 31, 2016 |
| Title |
ecT2 |
| Sample type |
SRA |
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| Source name |
Cortical neuronal culture
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
age: Embryonic day 18, 11 DIV
treatment: 1hr 1uM TTX inactivation
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| Treatment protocol |
Neurons were treated as described with 25mM KCl or 1uM TTX for 1hr. Media was removed and RNA was extracted immediately following treatment.
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| Growth protocol |
Neurons were removed from cortical tissue at embryonic day 18, and maintained in vitro for 11 days prior to experimental treatment. Each culture well contained ~250,000 cells.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from was extracted, DNase-treated, and purified (RNeasy, Qiagen). 2µg of total RNA underwent quality control (Bioanalyzer; all RIN values > 9.5) and library construction for polyA+ or polyA- RNA sequencing. For PolyA- RNA, an additional step (removal of ribosomal RNA with NEBNext rRNA depletion kit) occured prior to adapter ligation.
Directional polyA+ and polyA- RNA llibraries were constructed at HudsonAlpha Genomic Services Lab using NEBNext reagents (New England Biolabs) according to manufacturer's recommendations with minor modifications (including the use of custom library adapters and indexes). RNA libraries were quantified (Kapa Library Quant Kit, Kapa Biosystems), and underwent sequencing (25M total 50bp paired-end reads) on an Illumina sequencing platform (HiSeq2000).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
non-polyA RNA
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| Data processing |
Raw paired-end sequenced reads (as fastq format) underwent quality control (FASTQC) in Galaxy.
Low quality reads were filtered with the FASTX toolkit in Galaxy ("filter by quality"), with a quality cutoff of 20 and a minimun percentage of 90.
High-quality reads were aligned to the rat genome (rn5) in Galaxy using Tophat v1.4.0 (with custom settings –p 8 –r 175).
Genome-aligned sequenced reads for each sample were examined using SeqMonk (Babraham institute), using Ensemble v. 70 gene and feature annotations. For each sample, gene/transcript expression levels were determined by computing the fragments per kilobase of exon per million mapped reads (FPKM). For PolyA- RNA-seq, estimates of pre-TSS (1kb upstream of TSS), intronic, and post-TES (2kb downstream of TES) were also quantified for comparison.
Gene expression differences between groups (i.e., untreated versus KCl) were calculated in SeqMonk using all replicates for each group. Statistical significance was assessed using Student's t-tests and an false-discovery rate of 0.05.
Genome_build: rn5
Supplementary_files_format_and_content: Matrix with FPKM values for each sample (Columns) and each gene (rows). The first row contains column identifiers. The first column contains Refseq gene names or Region IDs.
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| Submission date |
Jan 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jeremy Jason Day |
| E-mail(s) |
jjday@uab.edu
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| Phone |
205-996-8960
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| Organization name |
UAB
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| Department |
Neurobiology
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| Lab |
Day
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| Street address |
1825 University Blvd
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| City |
Birmingham |
| State/province |
AL |
| ZIP/Postal code |
35294 |
| Country |
USA |
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| Platform ID |
GPL14844 |
| Series (2) |
| GSE64902 |
Extra-coding RNAs regulate neuronal DNA methylation and long-term memory formation [RNA-seq] |
| GSE64988 |
Extra-coding RNAs regulate neuronal DNA methylation dynamics |
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| Relations |
| BioSample |
SAMN03282182 |
| SRA |
SRX838726 |
| 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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