 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 04, 2012 |
| Title |
embryo wt rep2 |
| Sample type |
SRA |
| |
|
| Source name |
whole embryo
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: wild type
age: 8.5dpc
Sex: mixed
tissue: embryo
mean insert/fragment size: 147.794629
stddev insert/fragment size: 21.194443
sample type: genomic DNA after MeDIP
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
200 ng of genomic DNA was sonicated to an average size of 150bp. The sample was then purified using a Zymo DNA Clean and Concentrator-5 kit (according to the supplier’s instructions), and finally eluted in 20 μl of water (pre-heated to 50°C). The DNA was then end repaired: the following reaction (total vol = 25 μl) was prepared in 0.2 ml PCR tubes: • 200 ng of sonicated DNA (+ water if reqd so that the total vol = 18 μl) • 2.5 μl 10 X T4 DNA ligase buffer with 10mM ATP (NEB) • 1 μl of 10 µM dNTP mix • 1.25 μl of T4 DNA polymerase (from 3U/μl stock, NEB) • 1.25 μl of 1/5 dilution (in water) of Klenow DNA polymerase (from 5U/μl stock, NEB) • 1.25 μl of T4 Polynucleotide Kinase (from 10U/μl stock, NEB) Samples were gently mixed and incubate in a PCR machine for 30 minutes at 20°C, and then purified using a Zymo DNA Clean and Concentrator-5 kit (using 100 μl of binding buffer), and eluted in 13.5 μl of Elution buffer. The fragments were then A-tailed: the following reaction (total vol = 20 μl) was prepared in 0.2 ml PCR tubes: • DNA sample (13 μl) • 2 μl NEB Buffer 2 (from 10X stock) • 4 μl dATP (from 1 mM stock) • 1 μl Klenow exo (3’ to 5’ exo-) (from 5U/μl stock) The reaction was incubated for 30 minutes at 37°C in a PCR machine, and then purified a using Zymo DNA Clean and Concentrator-5 kit (using 100 μl of binding buffer), eluting in 14.5 μl of elution buffer. Illumina paired-end sequencing adaptors were ligated: the following reaction (total vol = 20 μl) was performed in 1.5 ml eppendorf tubes: • DNA sample (14 μl) • 4 μl of 5 X DNA ligase buffer (NEB, USA) • 1 μl of 1/10 dilution (in 1 X DNA ligase buffer) of standard Illumina PE adapter oligo mix • 1 μl NEB Quick ligase (NEB, USA) The reaction was incubated for 15 minutes at room temperature, and then purified using a Zymo DNA Clean and Concentrator-5 kit (using 100 μl of binding buffer), eluting in 25 μl of elution buffer (pre-heated to 50°C). 1 μl of this was kept aside as input, and another 1 μl analyzed on an Agilent Bioanalyzer to ensure ligation had occurred, with no significant left over adaptor contamination. 100ng of this ligated sample was transferred to a 1.5ml eppendorf tube and made up to 24 µl with water. This was denatured at 100°C for 10 minutes and immediately cooled on ice for 5 minutes. Then, 25 µl of chilled 2 X IP buffer (20 mM Na-Phosphate pH 7.0, 280 mM NaCl, 0.1 % Triton X-100) and 1 µl of 1/5 dilution (in 1 X IP buffer) of 5mC mAb was added, and the sample incubated overnight at 4°C with slow rotation. The next morning 1 µl of Dynabeads (Invitrogen,USA) were washed in 200 µl of 1 X IP buffer by capturing the beads with a magnetic rack (2 minutes). The buffer was then removed and discarded, the MeDIP sample was added to the Dynabeads, and then incubated for 6 hours at 4°C with slow rotation. Then, 150 µl of 1 X IP was added to the sample, the beads captured with a magnetic rack (for 2 minutes), supernatant discarded, and the beads washed with 200 µl 1 X IP buffer 3 more times (each time capturing the beads for 2 minutes). The beads were finally re- suspended in 50 µl Proteinase K digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5 % SDS) containing 0.5 µl of Proteinase K (10 mg/ml stock), and incubated for 1 hour at 55°C with rotation. The samples were briefly spun down, and the beads captured with a magnetic rack (2 minutes), and the supernatant transferred to 700 µl of binding buffer from the Zymo DNA Clean and Concentrator-5 kit and purified following the manufacturer’s instructions, finally eluting in 35 μl of water (pre-heated to 50°C). Then, the MeDIP and input samples were amplified as described below: • MeDIP sample (35 μl) or [1 μl input DNA + 34 μl water] • 5 μl of 10X Platinum Pfx polymerase buffer (Invitrogen, USA). • 2 μl of MgSO4 (from Platinum Pfx kit, Invitrogen, USA). • 2 μl of 10 mM dNTP mix • 0.8 μl of Platinum Pfx polymerase (Invitrogen, USA). • 2.5 μl of Illumina PCR primer 1.1 (from 10 μM stock) • 2.5 μl of Illumina PCR primer 2.1 (from 10 μM stock) Cycling conditions: a. 2 min/94°C s b. 12 cycles of: 15 s/94°C, 30 s/65°C, 30 s/68°C c. 5 min/68°C d. Hold/10°C PCR products were purified using the Qiagen PCR purification kit (using 650 μl buffer PB), and finally eluted in 30 μl EB buffer. MeDIP enrichment was checked by qPCR using the primers listed in Rakyan et al., 2008. Finally, approximately half of the PCR sample was run on a thin 3% Nusieve agarose gel, a band corresponding to 250 – 275 bp was excised and then purified using a Qiagen gel extraction kit as per the manufacturer’s instructions except that the gel was dissolved gel at room temp. for 10 minutes with vortexing. The sample was finally eluted in 30 μl of buffer EB and checked on an Agilent Bioanalyzer.
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
genomic DNA after MeDIP
pool of embryos
|
| Data processing |
Alignments: reads were aligned to mm9 using novoalign except for the sperm samples that were aligned using BWA. Details about aligner version and parameters used are stored in the headers of the BAM files. The stated values for mean insert size and standard deviation are derived from the paired-end alignments using Picard. In the case of the liver samples, which were single-end sequenced, mean and standard deviation of the inserts were estimated from Bioanalyzer measurements after size selection (10 size selections were carried out and pooled).
|
| |
|
| Submission date |
Oct 06, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Reiner Schulz |
| E-mail(s) |
[email protected]
|
| Organization name |
King's College London
|
| Department |
Medical & Molecular Genetics
|
| Lab |
Epigenetics
|
| Street address |
8th floor Guy's Tower
|
| City |
London |
| ZIP/Postal code |
SE1 9RT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE32687 |
Identification of maternal imprinting control regions (ICRs) in the post-implantation mouse embryo. |
|
| Relations |
| SRA |
SRX100670 |
| BioSample |
SAMN00738571 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM811235_WT2_dupmark.bam |
2.9 Gb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
