|
| Status |
Public on Aug 24, 2012 |
| Title |
Sample 71, MspI, Replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
Frontal cortex (BA10) of the brain - post-mortem sample
|
| Organism |
Homo sapiens |
| Characteristics |
enzyme digestion: 10 ng of native sheared DNA were subjected to 10 U of MspI digestion in a final volume of 20 µl for 12 h at 37ºC (50 fold surplus of the enzyme over the DNA substrate), followed by enzyme inactivation at 80ºC for 10 min.
age: 49 years
Sex: F
post mortem interval: 45
tissue: Frontal cortex (BA10) of the brain - post-mortem sample
|
| Treatment protocol |
No treatment performed on tissue prior to DNA extraction
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Micrococcus nuclease digestion was subjected to fragment genomic DNA to a median size of 500 bp and to reduce 3' hydroxyl end at the DNA fragments, where the latter is served as starting end for SMS. 5 µg of genomic DNA was treated with 1 U of micrococcal nuclease enzyme (NEB) and the reaction was stopped by adding access of EDTA (0.5M EDTA) in a time series. Samples with median fragment size of 500 bp were column purified with buffer PN (QIAquick Nucleotide Removal Kit columns, Qiagen). Glucosylation and control treatments were performed as described before (Online Methods) and 200ng of each glucosylated or non-glucosylated treated DNA was subjected to 10 U of restriction enzyme digestions respectively at 37 oC for 8h, and inactivated at 80oC for 20 minutes. 10 ng of each digested product, quantified by Quant-iTTM PicoGreen dsDNA Reagent Kit (Invitrogen), was then processed for Helicos sequencing. In brief 10ng of DNA was heat denatured at 95oC for 5 minutes prior to 3’ end labeling with 5 U of terminal transferase (NEB) in presence of 200 µmoles of dATP (Roche) and 5 mmoles of CoCl2 (NEB) in 20 µl reaction volume at 37oC for 1 h, inactivated at 70oC for 10 minutes. Fragments were biotinylated by repeating the terminal transferase enzymatic reaction step in presence of 100 µmoles of biotin labeled ddATP (Perkin Elmer) instead of dATP in a reaction volume of 30 µl. These processed samples were then sent to Helicos sequencing service facility (www.helicosbio.com; USA).
|
| |
|
| Library strategy |
MRE-Seq |
| Library source |
genomic |
| Library selection |
Restriction Digest |
| Instrument model |
Helicos HeliScope |
| |
|
| Description |
Three technical replicates of the same human brain DNA sample digested by MspI enzyme with (GluMspI) or without (MspI) prior glucosylation. Undigested DNA was used as a second reference. Data from all three runs were pooled for analysis after each run had been separately normalized using the corresponding number of non-target reads.
10 ng of native sheared DNA were subjected to 10 U of MspI digestion in a final volume of 20 µl for 12 h at 37ºC (50 fold surplus of the enzyme over the DNA substrate), followed by enzyme inactivation at 80ºC for 10 min.
MAY_fc1.ch02
Helicos single molecule sequencing (SMS)
|
| Data processing |
SMS reads were trimmed for leading “T” homopolymers, filtered for reads with a minimal length of 25 bases after trimming as well as for other standard Helicos quality metrics using a suite of Helicos tools available at: http://open.helicosbio.com/mwiki/index.php/Releases.
Alignments to the hg18 version of the human genome were conducted with indexDPgenomic
software freely available on the Helicos website (http://open.helicosbio.com/mwiki/index.php/Releases)
The sequence reads were aligned using a minimum normalized score of 4.3
Processed data file: Only uniquely-mapped reads were considered for the present analysis. Reads with a 5' coordinate < 3 bp from a target sequence (CCGG) were defined as target reads. Reads with a 5' coordinate > 200 bp away from a CCGG sequence (provided here as non-target reads) were used to normalize the read count in downstream analyses.
Genome_build: hg18
Supplementary_files_format_and_content: Processed data file: BED files of target reads (*coverage.txt) contain number of reads mapping to each CpG. BED files of non-target reads (NONTARGET.bed) contain number of reads mapping to the start coordinate of each non-target read.
Supplementary_files_format_and_content: Each sample has two results files: one for "target reads" (*coverage.bed)
Supplementary_files_format_and_content: and one for "nontarget reads" (*_NONTARGET.bed).
Supplementary_files_format_and_content: For target read files, the base position refers to the modifiable Cytosine
Supplementary_files_format_and_content: inside a restriction site (the second C of CCGG) which generated the read;
Supplementary_files_format_and_content: it is not the read start. For non-target read files, the base position
Supplementary_files_format_and_content: refers to the start of a read.
|
| |
|
| Submission date |
Aug 16, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Shraddha Pai |
| E-mail(s) |
shraddha.pai@utoronto.ca
|
| Organization name |
University of Toronto
|
| Street address |
160 College Street, Room 602
|
| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL14761 |
| Series (2) |
| GSE40158 |
5-hmC in the brain: abundance in synaptic genes and differences at the exon-intron boundary (MRE-Seq) |
| GSE40167 |
5-hmC in the brain: abundance in synaptic genes and differences at the exon-intron boundary |
|
| Relations |
| SRA |
SRX177672 |
| BioSample |
SAMN01120259 |
