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Sample GSM1063362

Query DataSets for GSM1063362

Status

Public on Jan 14, 2013

Title

MeDIP_SEQ PCP2 replica1

Sample type

SRA

Source name

Purkinje cells

Organism

Mus musculus

Characteristics

cell type: Purkinje cells
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

PC_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