|
| Status |
Public on Nov 02, 2016 |
| Title |
Rat rep 2 cortical neurons 6hr post-KCl stim |
| Sample type |
SRA |
| |
|
| Source name |
Long-Evans
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Long-Evans
tissue: E17 cortex, cultured, DIV7
time post-kcl stimulation: 6hr
replicate: 2
|
| Treatment protocol |
For KCl depolarization of neurons, neuronal cultures were first silenced overnight in culture media with 1 μM Tetrodotoxin (TTX) and 100 μM D-AP5. The following day samples were incubated for 0, 1 or 6 hours in 55 mM KCl prior to harvest.
|
| Growth protocol |
Mouse and rat neuronal cultures from three independent dissections were grown for RNA-seq experiments as outlined in the Methods section.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The libraries for RNA-Seq were constructed using the NEBNext Ultra Directional RNA library prep kit (for Illumina).
Strand-specific and single-end for RNA-Seq.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
For RNA-Seq of mouse and rat cortical cultures, strand-specific single-end reads of length 75 bp or shorter were trimmed at their 3' ends to 70 bp; those shorter than 70 bp (<0.2%) were discarded. The remaining 70-bp reads were mapped to the mm10 (mouse) or rn6 (rat) genome using BWA (v.0.7.8) allowing up to 2 mismatches. For mouse and rat respectively, the usual 20 and 21 chromosomal alignment targets were supplemented with their mitochondrial genomes plus ~8 and ~6 million short sequences. The latter targets represent all possible intragenic exonic sets from their respective RefSeq annotations whose junctions are spannable by 70-bp reads. Typically 80% of all reads in each mouse library (90% in rat) were mappable and ~50-80% of these aligned uniquely; nonuniquely mapped reads were discarded.
The exons for all transcripts assigned to a gene based on the RefSeq annotation for mouse (GRCm38/mm10; Dec. 2011) or for rat (RGSC 6.0/rn6; July 2014) were merged (unioned); along with the constructed library of exon-exon splice-junction sequences, these defined each gene's mature-RNA target region. The total number of bases of all reads that overlapped a gene's exonic region were divided by the total length of the region to yield an average read Density (coverage). Normalization of Densities to a standard of 10M 35-bp reads was effected by multiplying the raw Density by (10M/R)*(35/70), where R is the total number of uniquely mapped reads in a sample that did not overlap any RepeatMasker rRNA elements (RPKM units are proportional to Normalized Density units via RPKM = Density/0.35).
Genome_build: mm10
Genome_build: rn6
Supplementary_files_format_and_content: Standard bigWig-formatted files (.bw) for both mouse and rat RNA-seq samples represent bwa-aligned trimmed 70-bp reads piled up with single-base-pair resolution and then averaged over tiles of width 20bp. Each sample has its own pair of separate tracks for POS & NEG strands.
|
| |
|
| Submission date |
Aug 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Bulent Ataman |
| Organization name |
Harvard Medical School
|
| Department |
Neurobiology
|
| Lab |
Michael E. Greenberg
|
| Street address |
220 Longwood Ave. GB405
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL20084 |
| Series (1) |
| GSE78688 |
Activity-dependent transcriptional changes in human neurons |
|
| Relations |
| BioSample |
SAMN05574509 |
| SRA |
SRX2022803 |
