|
| Status |
Public on Apr 02, 2012 |
| Title |
AML_4 |
| Sample type |
SRA |
| |
|
| Source name |
Bone marrow
|
| Organism |
Homo sapiens |
| Characteristics |
cytogenetic: Normal karyotype (NK)
sample type: DNA of AML patient
medip antibody: 5- methyl cytosine (Diagenode.sa, MAb-081-100, Lot/batch DA-0016)
|
| Treatment protocol |
7.5 μl of monoclonal antibody (Diagenode 1μg/μl) is added to at least 4 μg of DNA for 2 hours at 4C with slow rotation. 40 µl of magnetic Dynabeads (Dynabeads M-280 sheep anti-Mouse IgG, Invitrogen) are prewashed with 1ml 1XIP buffer and collected with a magnetic rack. Dynabeads are added to the DNA sample and incubated for 2 hours at 4C with slow rotation. Dynabeads are collected using a magnet and washed with 750 µl 1X IP buffer for three times. To release the immunoprecipitated DNA, Dynabeads are resuspended in 200 µl digestion buffer and 5 µl of Proteinase Kinase (10mg/ml). The suspension is incubated for 2 hours at 55°C with shaking to avoid the sedimentation of the beads. After the incubation, the sample is divided in 2 separate tubes and purified by Zymo DNA Clean and Concentrator-5 kit (using 700 µl binding buffer) in 20 µl EB. 10 µl MeDIP and 10 ng of input DNA are subjected to limited PCR (LM PCR) using primers provided from Illumina for paired end library preparation. For size selection, 15 µl of MeDIP is run over 2% low melting temperature gel. MeDIP is excised from gel in the range of 250-300 bp and eluted in 30 µl EB using Qiagen Gel Extraction kit.
|
| Growth protocol |
Genomic DNA was extracted from AML by Qiagen kit, then the DNA sonicated by Sonic vibracel. The DNA frament will be blunt ended by adding klenow enzme and T4 polynucleotide kinase
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The quantified dsDNA MeDIP library was prepared for sequencing over Illumina flow cell. Preparation for sequencing includes firstly, DNA denaturation by NaOH solution and further diluted in HT1 buffer (tris buffer) to end into 4 PM concentration solution ready to be put over the flow cell. Secondly, the 'cluster generation' step, which is processed by adding a mixture of unlabelled nucleotides and DNA polymerase to the ssDNA library, which will attach to the primer sequences present inside the surface of the flow cell. The result is initiating the amplification of each fragment through a 'bridge amplification' reaction, which is unique to Illumina and subsequently generation of millions of clusters; each one represents a single fragment of MeDIP library. The average insert size of paired-end libraries was 200 bp and the standard deviation was of 20 bp. Illumina GAII, with the criteria of 1.4-pipeline software and 2.4-system control software (SCS) can produce 180.000-270.000 clusters/tile
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
monoclonal antibody to precipitate 5 methyl cytosine
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the human genome (build 36, March 2006) using the softwares 'maq' (http://maq.sf.net/) and 'Bowtie' (http://bowtie-bio.sourceforge.net/index.shtml).
DNA Methylation: Absolute DNAm states were inferred from alignment of raw reads using the Batman algorithm (Down et al., Nat Biotech 2008)
|
| |
|
| Submission date |
Apr 01, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jacek Dariusz Marzec |
| E-mail(s) |
j.marzec@qmul.ac.uk
|
| Organization name |
Barts Cancer Institute
|
| Department |
Centre for Haemato-Oncology
|
| Lab |
Cancer Genomics
|
| Street address |
Charterhouse Square
|
| City |
London |
| ZIP/Postal code |
EC1M 6BQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE28314 |
Genome-wide study of DNA methylation in AML by MeDIP-seq |
| GSE37022 |
Genome-Wide Analysis of Acute Myeloid Leukemia Reveals Leukemia-Specific Methylome and Subtype-Specific Hypomethylation of Repeats |
|
| Relations |
| SRA |
SRX093547 |
| BioSample |
SAMN00714421 |
| Named Annotation |
GSM700409_AML_4_rpm.WIG.gz |
