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Sample GSM2278979 Query DataSets for GSM2278979
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

Supplementary file Size Download File type/resource
GSM2278979_BA_rn-CTX_KCl-6hr_2_rn6_GNM-SPL_r70_span-20_NEG.bw 80.0 Mb (ftp)(http) BW
GSM2278979_BA_rn-CTX_KCl-6hr_2_rn6_GNM-SPL_r70_span-20_POS.bw 80.2 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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