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Sample GSM1063329 Query DataSets for GSM1063329
Status Public on Jan 14, 2013
Title Cerebellum wt male 2
Sample type SRA
 
Source name Cerebellum
Organism Mus musculus
Characteristics cell type: Cerebellum
strain: C57/Bl6
age: 6 weeks
genotype: wt
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Data processing 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).
 
Submission date Jan 14, 2013
Last update date May 15, 2019
Contact name Marian Mellen
Organization name The Rockefeller University
Street address 1230 York Avenue
City New York
ZIP/Postal code 10065
Country USA
 
Platform ID GPL13112
Series (1)
GSE42880 MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system
Relations
SRA SRX216821
BioSample SAMN01886292

Supplementary file Size Download File type/resource
GSM1063329_Cb_ND1_2_2_RNASEQ_CTTGTA_L007_R1_001.accepted_hits.bam.wig.gz 40.5 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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