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| Status |
Public on Jan 14, 2013 |
| Title |
5hmC-Seq ND1 replica 2 |
| Sample type |
SRA |
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| Source name |
Cerebellar Granule cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Cerebellar Granule cells
strain: C57/Bl6
age: 8-12 weeks
genotype: wt
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| 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
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
5hmC-Seq
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| 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.
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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 |
SRX216845 |
| BioSample |
SAMN01886316 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1063353_ND1_A_10032011_PLUS_GGCTAC_L002_R1_001.bowtie.bam.wig.gz |
216.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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