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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jan 14, 2013 |
Title |
sept4 TRAPSeq male 1 |
Sample type |
SRA |
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Source name |
Bergmann glia
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Organism |
Mus musculus |
Characteristics |
cell type: Bergmann glia strain: C57/Bl6 age: 8 weeks library strategy: TRAP-Seq rna subtype: polysomal RNA
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Extracted molecule |
total RNA |
Extraction protocol |
Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 μg/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 × g, 4 °C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 × g to pellet insoluble material. At this point, 30 μL of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4°C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 μg/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA per IP or input sample were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturers’ instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 μg of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2000 |
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Description |
polysomal RNA
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Data processing |
Library strategy: TRAP-Seq Data were separatedly aligned to the mouse genome (mm9) downloaded from UCSC. TopHat software (version 1.3.1) was used for processing reads. Segment size was set to 25bp with two mismatches to the reference allowed, and the minimum anchor size was set to 10bp with no mismatches allowed. differentially expressed genes were identified by performing a negative binomial test using the DESeq package (Anders and Huber, 2010) of R/Bioconductor (Gentleman et al., 2004).
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Submission date |
Jan 14, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Marian Mellen |
Organization name |
The Rockefeller University
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Street address |
1230 York Avenue
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City |
New York |
ZIP/Postal code |
10065 |
Country |
USA |
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Platform ID |
GPL13112 |
Series (1) |
GSE42880 |
MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system |
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Relations |
SRA |
SRX216825 |
BioSample |
SAMN01886296 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1063333_Marian_sept4_1_1_1_IPB_09302011_F_2011_10_19.accepted_hits.bam.wig.gz |
19.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
Processed data provided as supplementary file |
Raw data are available in SRA |
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