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Sample GSM2109046 Query DataSets for GSM2109046
Status Public on May 27, 2016
Title Top1_cKO_Topot_Rep3
Sample type SRA
 
Source name Cortical neurons
Organism Mus musculus
Characteristics strain: C57BL6
genotype: Top1cKO
cell type: cortical neurons
treatment: Topotecan
Treatment protocol Lentivirus from the pLenti-CaMKIIα-tdTomato and pLenti-CamKIIα-tdTomato-P2A-CRE vectors were prepared by the UNC Lentiviral Core. Lentiviral Top1 shRNA was generated as previously described [22]. Briefly, cortical neurons were transduced at DIV 3 with lentivirus at a multiplicity of infection of at least two to maximize the number of transduced cells (around 85-90% transduction efficiency). Media containing lentivirus was removed 24 hours later and replaced with conditioned media. The CaMKIIα promoter limited tdTomato expression to neurons and was detectable without antibody amplification 3-4 days post transduction. Neurons were then treated at DIV 15 with vehicle (0.003% DMSO, Neurobasal medium) or 300 nM topotecan (Molcan Corporation; in 0.003% DMSO, Neurobasal medium) and harvested 3 days later.
Growth protocol Embryonic day E13.5-15.5 mouse cortical neuron cultures were prepared from adult C57BL6/J wild-type females as described in Huang et al (Nature, 2012).
Extracted molecule polyA RNA
Extraction protocol RNA was isolated with the RNeasy plus mini kit (Cat. #74134, Qiagen).  RNA yield and quality was determined with a Nanodrop 1000 Spectrophotometer (Thermo Scientific).  Samples were further assessed for quality using either an Agilent Bioanalyzer 2100 or TapeStation 2200 to obtain a RNA integrity number (RIN).  RIN values exceeding 7 were used for sequencing
RNA samples were used to generate and barcode cDNA libraries using the TruSeq RNA Library Preparation Kit at the UNC High Throughput Sequencing Facility.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing RNA-seq reads were filtered using TagDust
Reads were aligned to the reference mouse genome (mm9) with TopHat using default parameters
Reads aligning to rRNA genes were removed
Transcript abundance was estimated by computing RPKM and raw counts using RefSeq gene models aggregated by gene symbol
Genome_build: mm9
Supplementary_files_format_and_content: Matrix of raw counts for each sample and each RefSeq gene. Cntnap2 gene model was adjusted to reflect the neuronal isoform
 
Submission date Apr 05, 2016
Last update date May 15, 2019
Contact name Mark Zylka
Organization name University of North Carolina - Chapel Hill
Street address 5109D Neuroscience Research Building
City Chapel Hill
State/province NC
ZIP/Postal code 27599
Country USA
 
Platform ID GPL17021
Series (1)
GSE79951 Topoisomerase 1 Regulates Gene Expression in Neurons through Cleavage Complex-Dependent and -Independent Mechanisms
Relations
BioSample SAMN04613918
SRA SRX1681549

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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