 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 20, 2013 |
| Title |
EOS-stable-thymus_RRBS |
| Sample type |
SRA |
| |
|
| Source name |
EOS stable FoxP3+ T-cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Thymus
cell type: CD4+GFP+CD103+CD38NEG
genotype/variation: BAC-tg Foxp3/GFP-Cre (Bluestone)
|
| Treatment protocol |
No treatment involved.
|
| Growth protocol |
Mouse spleen and thymus tissues were harvested and minced into small pieces. Treg cells were fragile during staining, and we found that it was important to disaggregate LNs rapidly (by passing once through a 40 um mesh) and stain quickly using short incubation times (10 min on ice). The stained cells were sorted immediately and proceeded for DNA isolation. At least 250,000 cells were sorted for the methylation analysis.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
RRBS was performed according to a previously published protocol (Gu et al. 2010; Meissner et al. 2008) with minor modifications.
For each sample, 1 ug genomic DNA was first digested overnight using 20 units of MspI (New England Biolabs, NEB). The digested DNA was end-repaired and adenylated in a 20 ul reaction consisting of 10U of Klenow fragments (exo-, Enzymatics, MA, USA), and 2 ul premixed nucleotide triphosphates (1 mM dGTP, 10 mM dATP, 1 mM methylated dCTP). The reaction was incubated at 30 C for 30 min followed by 37 C for an additional 30 min. The methylated Illumina adapters were ligated with adenylated DNA fragments in a 20 ul reaction containing 1 ul concentrated T4 ligase (Enzymatics) at room temperature for 15 minutes. The ligation products were gel-selected for fragments with insertion sizes of 40 to 120 bp and 120 to 220 base pairs as previously suggested (Gu et al. 2010; Meissner et al. 2008). Bisulfite treatment of the sequencing library was conducted using the EZ DNA methylation kit (Zymo Research, Irvine, CA) according to manufacturer's protocol. The final libraries were generated by PCR amplification of 5 um of bisulfite-converted template using PfuTurbo Cx Hotstart polymerase and Illumina pair-end PCR primers (PE1.0 and PE2.0).
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
DNA methylation analysis using bisulfite sequencing
|
| Data processing |
Base-calling: CASAVA version 1.8
Alignment: Bowtie
Sequence reads were trimmed for adaptor sequence and low-quality sequence in the both end.
- C->T converted sequence reads were mapped to C->T converted strands of mm9 genome using bowtie V0.12.7 with -n 2 -e 150 -k 10 --chunkmbs 250 --maxbts 800 --best -t -S
- For each read, the best alignment was chosen based on the number of mismatches
- Filtered out reads that were mapped to multiple locations
- Filtered out reads with percent identity of alignment < 95%
- For each CpG, methylation fraction (Beta value) was calculated by the number
Genome_build: mm9
Supplementary_files_format_and_content: bed files
The 7 columns in the bed file are:
1. Chromosome
2. Start
3. End
4. Number of reads
5. Methylation fraction
6. Strand direction
7. Number of methylated reads
|
| |
|
| Submission date |
Feb 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Huidong Shi |
| E-mail(s) |
[email protected]
|
| Phone |
706-721-6000
|
| Organization name |
Augusta University
|
| Department |
Georgia Cancer Center
|
| Lab |
2125 K
|
| Street address |
1120 15th Street, CN2138
|
| City |
Augusta |
| State/province |
GA |
| ZIP/Postal code |
30912 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE44380 |
An inherently bi-functional subset of Foxp3+ Treg/T-helper cells is controlled by the transcription factor Eos |
|
| Relations |
| SRA |
SRX244143 |
| BioSample |
SAMN01924265 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1084107_EOS-stable-thymus_RRBS.cg.bed.gz |
16.4 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
