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Sample GSM1063364 Query DataSets for GSM1063364
Status Public on Jan 14, 2013
Title MeDIP_SEQ SEPTIN4 replica1.1
Sample type SRA
Source name Bergmann glia
Organism Mus musculus
Characteristics cell type: Bergmann glia
strain: C57/BL6
age: 8-12 weeks
Extracted molecule genomic DNA
Extraction protocol Isolation and sorting was performed as described in (Kriaucionis and Heintz, 2009). Briefly, cerebella were dissected as described above and homogenized in homogenization medium (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail (Roche) using loose (A) and tight (B) glassglass dounce.Homogenate was supplemented with 50% iodixanol (OptiPrep, Axis-Shield, Scotland), 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8; and laid on a 29% iodixanol cushion. Cerebella from 12 mice (6 males and 6 females) were used for PCs, 6 (3 males and 3 females) for BGs and 4 (2 males and 2 females) for GCs. Nuclei were pelleted by centrifugation 30 min, 10,000 g, 4 °C in swinging bucket rotor (SW41) in a Beckman Coulter XL-70 ultracentrifuge. The nuclear pellet was resuspended in homogenization buffer and co-stained with DyeCycle Ruby (Invirogen) to 20 μM final concentration. Nuclei were sorted in a BD FASCAria (BD Biosiences, San Jose, CA, USA) cell sorter using 635 nm and 488 nm excitation lasers and by gating with two parameters: high GFP signal (compared to wt mice) indicating bacTRAP positive cells and lowest signal for DC Ruby, indicating singlets. Nuclei were fast frozen in liquid nitrogen after sorting and kept at -80 C until analysis.
MeDIP was done as described in (Weber et al., 2005) with the indicated modifications. 1-0.5 μg of DNA was used for each experiment. Sonicated DNA was end-repaired following by ligation to Illumina paired end sequencing adapters (Illumina, PE‐ 102‐ 1003). Enrichment was done using anti-methylC antibody (Eurogentech, BI-MECY-0100), following by amplification with Illumina primers and size selection on an agarose gel. Input samples were produced for each cell types in both procedures
Library strategy MeDIP-Seq
Library source genomic
Library selection 5-methylcytidine antibody
Instrument model Illumina Genome Analyzer II
Description BG_mC_32M.bedGraph.gz
Data processing Reads were aligned to mm9 mouse genome assembly using Bowtie v0.12.7 (-m1 --best). (-m1 --best).
Further analysis was done using Bioconductor v2.9 using packages chipseq, biomaRt, rtracklayer, MEDIPS and custom scripts.
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 GPL9250
Series (1)
GSE42880 MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system
SRA SRX216856
BioSample SAMN01886327

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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