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Sample GSM1608024 Query DataSets for GSM1608024
Status Public on Jul 03, 2020
Title 11Dec06_FF
Sample type SRA
Source name microglia
Organism Mus musculus
Characteristics tissue: optic nerve
age: 3 months
gender: F
genotype/variaton: Nf1 flox/flox
strain: C57BL/6
Treatment protocol Tissue was dissociated, enriched by Percoll gradient, and cells were flow sorted.
Growth protocol not applicable
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from snap-frozen flow sorted cell pellets using TRIzol-chloroform extraction. RNA was resuspended in 10µl Ambion Nuclease-free water (Life Technologies) and DNAse treated (Ambion TURBO DNA-free Kit)
For cDNA synthesis, we added 5µl of isolated RNA into the Ovation® RNA-Seq method.  500ng cDNA was fragmented using covaris shearing and processed for Illumina library construction with the Illumina paired-end LT indexing protocol as previously published (Mardis et al., 2009; Govindan et al., 2012). Each library was sequenced on the Illumina HiSeq, generating between 15-22Mbp per lane.
RNA-Seq (300-500bp size fractionation): cDNA samples were constructed into Illumina libraries according to the manufacturer’s protocol (Illumina Inc, San Diego, CA) with the following modifications: 1) DNA was fragmented using Covaris S2 DNA Sonicitor using the following condtions: Duty Cycle: 5, Intensity: 4, Cycles/Burst: 200, Time: 90sec (Covaris, Inc. Woburn, MA). Fragment sizes ranged between 100 and 500bp. 2) Illumina adapter-ligated DNA was amplified in a single 50ml PCR for five cycles. 3) Solid Phase Reversible Immobilization (SPRI) bead cleanup was used to purify the PCR and select for 300-500bp fragments. We added 0.8 volumes of the AmpureXP solution (preformulated with polyethylene glycol and sodium chloride) to size select DNA molecules greater than 300 bp. The size-fractioned cDNA was washed three times with 750 µl of 70% ethanol, the beads were dried, and the cDNA library was eluted off the beads by adding 20µl 10 mM Tris-HCl (pH 8.0). The size-fractioned libraries were assayed using the Agilent BioAnalyzer High Sensitivity DNA chips.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description FF biological replicate 4
Data processing Basecalls performed using Illumina Real-Time Analysis (RTA) version ( for MiSeq) followed by demultiplexing of the index sequence using CASAVA 1.8.2.
SPIA adapter sequence aligned and trimmed from raw sequence reads using FAR 2.17 with command line parameters : --adapter CTTTGTGTTTGA --trim-end left --adaptive-overlap yes --format fastq --write-lengthdist yes --nr-threads 4 --min-overlap 7 --max-uncalled 150 --min-readlength 25
Aligned with TopHat 2.0.4, Bowtie 2.0.0-beta7 and Ensembl 67 annotation (-G param)
FPKM calculated per sample with Cufflinks 2.0.2 and Ensembl 67 annotation with -G param
Genome_build: mm9
Supplementary_files_format_and_content: FFC-FF_gene_fpkm.tsv a tab delimited file with columns for gene ID and the normalized expression values per sample name
Submission date Feb 11, 2015
Last update date Jul 03, 2020
Contact name Winnie W Pong
Organization name Washington University School of Medicine
Department Department of Neurology
Lab David H Gutmann Laboratory
Street address 660 S. Euclid Avenue
City St Louis
State/province MO
ZIP/Postal code 63110
Country USA
Platform ID GPL13112
Series (1)
GSE65865 Low-grade glioma microglia
BioSample SAMN03344066
SRA SRX876794

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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