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Sample GSM1939694

Query DataSets for GSM1939694

Status

Public on Apr 22, 2016

Title

ca1d_1 (Hipposeq)

Sample type

SRA

Source name

Dorsal CA1 pyramidal cells

Organism

Mus musculus

Characteristics

cell type: CA1 pyramidal cell
location: CA1
tissue: hippocampus

Extracted molecule

total RNA

Extraction protocol

Manual sorting to purify for fluorescent neurons from microdissected slices was performed according to previous methods (Hempel et al., 2007).
Total RNA was isolated from each sample using PicoPure RNA Isolation kit (Life Technologies, Frederick, MD) including the on-column RNAseq-free DNAseI treatment (Qiagen, Hilden, Germany) following the manufacturers’ recommendations. Eluted RNA (11 ul) was dried in a speed vac to approximately 2-4 ul. ERCC control RNAs (Life Technologies) were added using 1 ul of 1:100,000 dilution for every 50 cells. cDNA was amplified from this input material using Ovation RNA-seq v2 kit (NuGEN, San Carlos, CA). Approximately half of the resulting cDNA was used to make the sequencing libraries using the Ovation Rapid DR Multiplexing kit (NuGEN).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Reads for each library were mapped using TopHat v2.0.6 9. The following options were used: “--num-threads 8 --GTF mouseGtf.gtf” , where mouseGtf.gtf reflects the concatenated Ensembl NCBIM37 transcript annotation file and the annotated ERCC spike-in controls
For FPKM-based quantification and differential expression, the annotated mouseGtf.gtf was used in Cuffdiff v2.1.1 using the accepted_hits.bam files for all replicates. The following options were used: “--frag-bias-correct mouseFa.fa --mask-file mouseMask.gtf --max-bundle-frags 10000000 --num-threads 8 --multi-read-correct --no-effective-length-correction”, where mouseFa.fa is the Ensembl NCBIM37 reference FASTA and mouseMask.gtf is a mask file that ignores all alignments corresponding to genes annotated in mouseGtf.gtf annotated as tRNA, rRNA, or snRNA; in addition, Nat8l, Psd2, Xist, Gm15459, and Gm10335 were masked.
For count-based quantification and differential expression, aligned reads were processed by HTSeq using the mouseGtf.gtf and the following options: "--format=bam --stranded=no".
Processed data were analyzed in the R environment using a combination of cummeRbund v3.0 (http://compbio.mit.edu/cummeRbund/), DESeq2, and custom scripts.
Genome_build: mm9
Supplementary_files_format_and_content: Tab delimited files containing FPKM values and pairwise DE calls, as well as count values

Submission date

Nov 13, 2015

Last update date

May 15, 2019

Contact name

Mark Steven Cembrowski

Organization name

University of British Columbia

Department

Cellular and Physiological Sciences

Lab

Cembrowski