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| Status |
Public on Jul 03, 2020 |
| Title |
11Dec06_FF |
| Sample type |
SRA |
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| Source name |
microglia
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| Organism |
Mus musculus |
| Characteristics |
tissue: optic nerve
age: 3 months
gender: F
genotype/variaton: Nf1 flox/flox
strain: C57BL/6
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| Treatment protocol |
Tissue was dissociated, enriched by Percoll gradient, and cells were flow sorted.
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| Growth protocol |
not applicable
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| 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
FF biological replicate 4
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| Data processing |
Basecalls performed using Illumina Real-Time Analysis (RTA) version 1.13.48.0 (1.13.56.0 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
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| Submission date |
Feb 11, 2015 |
| Last update date |
Jul 03, 2020 |
| Contact name |
Winnie W Pong |
| Organization name |
Washington University School of Medicine
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| Department |
Department of Neurology
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| Lab |
David H Gutmann Laboratory
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| Street address |
660 S. Euclid Avenue
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| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
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| Relations |
| BioSample |
SAMN03344066 |
| SRA |
SRX876794 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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