|
| Status |
Public on Dec 01, 2014 |
| Title |
h_BrainE5_MBD_M |
| Sample type |
SRA |
| |
|
| Source name |
Hippocampus
|
| Organism |
Homo sapiens |
| Characteristics |
individual: D20
gender: Male
age: 40
neuropathological status: Control
tissue: Hippocampus
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted and sheared to approximately 400 bp using sonication and then ligated to Illumina's adapters. Methylated DNA was purified by two step MBD Affinity Purification as described in Skene et al., 2010.
Libraries were prepared as follows using enzymes from New England Biolabs (NEB). End Repair was carried out by incubating fragmented DNA with T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase for 30 minutes at 20 C. The blunt, phosphorylated ends were treated with Klenow fragment (exo-) and dATP for 30 minutes at 37 C to yield a protruding 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. Ligated DNA was ethanol precipitated and then reconstituted in running buffer prior to MAP-purification. Purified DNA was PCR amplified with Illumina primers for 10-12 cycles. Library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated by gel extraction. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on a Genome Analyzer following the manufacturer's protocols. Sequencing was performed at the Wellcome Trust Sanger Institute; Hinxton, Cambridge, UK.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Library strategy: MBD-Seq
Library selection: MeCP2 protein methyl-CpG binding domain
Sequenced reads were mapped to hg18 genome using MAQ with parameters -n 2 -m 0.001 -e 70 -a 250
Reads with a poor mapping score were filtered out using bamtools with the paramater -mapQuality ">=20"
BAM files were converted into wig format using bedtools
Genome_build: hg18
Supplementary_files_format_and_content: Merged files containing the combined read data for all replicates for that sample. Columns represent: chromosome, start position, stop position & read_coverage. A single header row provides additional information for the browser.
|
| |
|
| Submission date |
Sep 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Rob S Illingworth |
| E-mail(s) |
[email protected]
|
| Phone |
01316519640
|
| Organization name |
The University of Edinburgh
|
| Department |
Centre for regenerative Medicine
|
| Lab |
Illingworth
|
| Street address |
Centre for Regenerative Medicine
|
| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH16 4UU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL9115 |
| Series (1) |
| GSE50960 |
Assessing inter- and intra-individual DNA methylation profiles in human brain samples |
|
| Relations |
| BioSample |
SAMN02358409 |
| SRA |
SRX352424 |
