 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 10, 2015 |
| Title |
RRMAB-Seq_shTdg |
| Sample type |
SRA |
| |
|
| Source name |
ESC E14
|
| Organism |
Mus musculus |
| Characteristics |
treatment: shRNA Tdg
cell type: embryonic stem cells
developmental stage: E14
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using DNeasy Blood and Tissue kit (Qiagen).
1 μg of mESC genomic DNA (plus 1 ng of E. coli DH10B genomic DNA) was incubated with 4U of M.SssI CpG Methyltransferase (NEB), supplemented with 160 μM final S-Adenosyl methionine for 2 h at 37°C. The incubation was repeated 3 times to maximize CpG methylation. For whole-genome MAB-Seq, methylated DNA was sheared using a Bioruptor® Twin (Diagenode) for 2 runs of 15 cycles [30 s “ON,” 30 s “OFF”] at high power setting. For Reduced Representation MAB-Seq (RRMAB-Seq), methylated DNA was digested for 4h at 37°C with 200U of MspI restriction endonuclease (NEB).
Fragmented/digested DNA was then end-repaired, dA-tailed, and ligated to methylated adapters, using the Illumina TruSeq DNA Sample Prep Kit, following manufacturer instructions. Adapter-ligated DNA, was treated for additional 3 cycles with M.SssI enzyme to methylate the unmodified cytosines introduced by the end-repair step. DNA was loaded on an EGel Size select 2% agarose pre-cast gel (Invitrogen), and a fraction corresponding to fragments ranging from 180bp to 350bp was recovered. Purified DNA was then subjected to bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen). Bisulfite-converted DNA was finally enriched by 15 cycles of PCR using Pfu Turbo Cx HotStart Taq (Agilent).
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiScanSQ |
| |
|
| Description |
DNA was treated with M.SssI and EpiTect Bisulfite Kits (Qiagen)
library strategy: MAB-Seq and RRMAB-Seq
|
| Data processing |
Basecalls performed using CASAVA version 1.8
Nucleotide positions with a quality score behind 20 (Phred33 scale) were trimmed using the fastx_trimmer tool from the FASTX Toolkit
Reads were subjected to adapter clipping using the fastx_clipper tool from the FASTX Toolkit, and reads shorter than 35nt were discarded
Reads were aligned to the mouse genome assembly using Bismark v0.10.1 using the default parameters
Methylation status was peformed using Methylation Extractor tool of Bismark.
The 5fcaC sites were determined by using a binomial test for each cytosine = pbinom(x, N, p) where N is the sequencing depth at the C residue, p is the fail-rate of the M.SssI treatment, as estimated on E. coli genome, and x is the number of times that the C residue was sequenced as T. Only the cytosines with a coverage > 10 and a p-value < 0.01 were considered for further analysis.
Genome_build: mm9
Supplementary_files_format_and_content: bed files with percentage of fcaC
|
| |
|
| Submission date |
Mar 06, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Francesco Neri |
| E-mail(s) |
francesco.neri@unito.it
|
| Organization name |
University of Torino
|
| Street address |
Via Nizza 52
|
| City |
Torino |
| State/province |
Italy |
| ZIP/Postal code |
10126 |
| Country |
Italy |
| |
|
| Platform ID |
GPL16173 |
| Series (2) |
| GSE55658 |
Single base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals the actual promoter DNA methylation dynamics in embryonic stem cells [MAB-Seq] |
| GSE55660 |
Single base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals the actual promoter DNA methylation dynamics in embryonic stem cells |
|
| Relations |
| BioSample |
SAMN02675487 |
| SRA |
SRX482058 |
| Named Annotation |
GSM1341315_RRMAB-Seq_shTdg.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1341315_RRMAB-Seq_shTdg.bed.gz |
89.5 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
