|
| Status |
Public on Nov 05, 2013 |
| Title |
2 dpi MGPC, apobec2a,2b Mo_Sample 12847 |
| Sample type |
SRA |
| |
|
| Source name |
Muller glia
|
| Organism |
Danio rerio |
| Characteristics |
strain: gfap:gfp
treatment: Injured, apobec2a,2b knockdown
time: 2 days post injury
tissue: retina
|
| Growth protocol |
Zebrafish were kept at 26-28 C on a 14/10 hr light/dark cycle.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Zebrafish retinas were collected in Leibovitz’s L15 medium, treated for 15 min with 1 mg/mL hyaluronidase at room temperature, and then dissociated in 0.01% (vol/vol) trypsin with frequent trituration. A single-cell suspension was confirmed by microscopy and target cell (GFP+ or GFP, lissamine morhpolino+) populations were isolated by FACS. gDNA was isolated from FACS purified cells using the Purelink Genomic DNA Mini Kit (Invitrogen) according to the manufacturer’s recommendations. The time between the harvest and DNA isolation of any sample did not exceed 3 hrs. The quantity of dsDNA from each sample was measured using a Qubit 2.0 Fluorometer.
gDNA samples were digested for 4 hrs with MspI, purified, and end-repaired and A-tailed with Klenow fragment 3’->5’ exo- (NEB). The samples were then purified and resuspended in Truseq DNA Sample Preparation (Illumina) Resuspension Buffer and submitted to a second round of adenylation followed immediately by ligation to barcoded Illumina adapters using the reagents and instructions of the Illumina TruSeq DNA Sample Preparation kit. The samples were then size selected on a 3% Nusieve 3:1 TBE gel and run at 5V/cm. Fragment sizes ranging from 160-340 bp (corresponding to 40-220 bp without adapter sequence) were excised from the gel and purified. Samples were then bisulfite converted using the EZ DNA Methylation-Direct Kit (Zymo), followed by amplification using PfuTurbo Cx Hotstart DNA Polymerase (Agilent) using the primers provided in the Illumina TruSeq DNA Sample Preparation kit. The libraries were then submitted to a second step of size selection on a 3% Nusieve 3:1 TBE gel, quality checked on an Agilent 2100 Bioanalyzer, DNA 1000 Tapestation and dsDNA quantified with a Qubit 2.0 Fluorometer. 50 ng of the final libraries were submitted for sequencing at the University of Michigan DNA Sequencing Core on an Illumina HiSeq 2000.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Bisulfite treated, MspI digestion
apobec2a,2b morpholino was delivered at the time of injury (6 x 0.7 μl of 0.25mM morpholino injections per eye) by using a Hamilton syringe. Morpholino delivery to cells was facilitated by electroporation. 6 additional lesions per eye were performed following electroporation.
|
| Data processing |
Basecalls performed with CASAVA version 1.8.2
Sequences were trimmed to remove PCR primers, adapter sequences and low-quality base calls (score <20) with TRIM_GALORE version 0.2.1
Reads (100b paired for MG and 4dpi mSC samples; 50b single for 2 dpi samples) were aligned to the Zebrafish reference genome using BISMARK version 0.7.3. (parameters -n 1 -l 28)
The BISMARK script METHYLATION_EXTRACTOR was used to extract methylation calls for every Cytosine.
CpG context files were processed using the methylKit R package version 0.5.4
Genome_build: Zv9
Supplementary_files_format_and_content: Methylation call files were generated using the methylKit R package's read() function.
Supplementary_files_format_and_content: CpG methylation calls
Supplementary_files_format_and_content: CHG methylatiion calls
Supplementary_files_format_and_content: CHH methylation calls
|
| |
|
| Submission date |
Sep 10, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ana Rodrigues Grant |
| E-mail(s) |
anapcr@umich.edu
|
| Organization name |
University of Michigan
|
| Department |
Computational Medicine & Bioinformatics
|
| Street address |
100 Washtenaw Avenue
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
| |
|
| Platform ID |
GPL14875 |
| Series (1) |
| GSE50717 |
Genome-scale DNA methylation maps of Zebrafish Muller glia during retina regeneration |
|
| Relations |
| BioSample |
SAMN02351662 |
| SRA |
SRX347394 |
