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Sample GSM1063349 Query DataSets for GSM1063349
Status Public on Jan 14, 2013
Title 5hmC-Seq PCP2 replica 2
Sample type SRA
Source name Purkinje cells
Organism Mus musculus
Characteristics cell type: Purkinje cells
strain: C57/Bl6
age: 8-12 weeks
genotype: wt
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.
5hmC was pulled down as described (Song et al., 2010). After purification DNA was amplified as described in TruSeq DNA Sample kit. 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) following by amplification with Illumina primers. Input samples were produced for each cell types in both procedures. 5hmC enriched were then sequenced using Illumina platform obtaining more than 50 x10-6, 36- bp single-end reads per sample. Reads were aligned to mm9 mouse genome assembly using Bowtie v0.12.7 (-m1 --best).Two biological replicas were done for each of the cell type
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2000
Description 5hmC-Seq
Data processing Library strategy: 5hmC-Seq
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 GPL13112
Series (1)
GSE42880 MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system
SRA SRX216841
BioSample SAMN01886312

Supplementary file Size Download File type/resource
GSM1063349_PCP2_6.2_10032011_PLUS_GATCAG_L005_R1_001.bowtie.bam.wig.gz 143.6 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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