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| Status |
Public on May 23, 2019 |
| Title |
MeDIPseq.ID4eGFP_Dim_Replicate1 |
| Sample type |
SRA |
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| Source name |
Spermatogonia
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| Organism |
Mus musculus |
| Characteristics |
strian: C57BL/6
tissue: testis
age: P6
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| Growth protocol |
Cells were subjected to flow cytometry using BD FACS Aria. Propidium iodide (PI) was added to discriminate dead cells . Positive ID4-eGFP epifluorescence was determined by comparison to testis cells from testes of wild-type mice lacking the P6 Id4-eGfp transgene. The gating area of eGFP positive was subdivided into thirds to define the ID4-eGFP+ subsets as being Dim (lower third) or Bright (upper third) by fluorescent intensity.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After FACS sorting, each aliqout (~50,000) of fresh cells was pelleted and re-suspended in lysis buffer (50mM Tris pH 8.0, 10 mM EDTA, 100 mM NaCl, 1% SDS, 0.5 mg/ml proteinase K) and incubated at 55°C for 5h. Genomic DNA was isolated using Phenol:Chloroform:Isoamyl Alcohol (Invitrogen #15593031), and sheared using a Bioruptor (Diagenode UCD-200). 10% raw sheared DNA was retained to serve as an input control. Samples were end-repaired in 1x T4 DNA ligase buffer, 0.4mM dNTP mix, 2.25U T4 DNA polymerase, 0.75U Klenow DNA polymerase and 7.5U T4 polynucleotide kinase for 30 min at 21-25°C, then A-tailed in 1x NEB buffer 2, 0.4mM dNTPs and 3.75U of Klenow(exo-) for 30 min at 37°C, and then ligated in 1x rapid DNA ligation buffer, 1mM Illumina PE adapters and 1,600U DNA ligase for 1-8 h at 21-25°C. Samples were denatured at 95°C for 10 min, then transfered immediately to ice to prevent re-annealing. 0.2pM λ-DNA fragments (50% methylated) were used as a spike-in control. MeDIP on purified adapter-ligated DNA with spike-in was performed in 0.1 M Na2HPO4/NaH2PO4, 35μl of 2 M NaCl, 2.5 μl of 10% Triton X-100 and 1μl of anti-methylcytidine antibody (1 mg/ml Diagenode # MAb-081-100) overnight. DNA-IgG complexes were captured by protein A/G agarose beads. DNA was extracted by phenol:chloroform:isoamyl alcohol (25:24:1, v/v).
IPed DNA were end-repaired in 1x T4 DNA ligase buffer, 0.4 mM dNTP mix, 2.25U T4 DNA polymerase, 0.75U Klenow DNA polymerase and 7.5U T4 polynucleotide kinase for 30 min at 21-25°C, then A-tailed in 1x NEB buffer 2, 0.4 mM dNTPs and 3.75U of Klenow(exo-) for 30 min at 37°C and then ligated in 1x rapid DNA ligation buffer plus 1mM Illumina PE adapters and 1,600U DNA ligase for 1-8h at 21-25°C. Ligated fragments were amplified using dual-indexed primers for Illumina (NEB #E7600S) for 8-10 PCR cycles. DNA was purified with 1.8x volume Ampure XP DNA purification beads (Beckman Coulter #A63881) between each step. Fragment length was again checked by Bioanalyzer (Aglient Technology), and DNA concentration was determined using the Qubit dsDNA HS Assay Kit (Invitrogen #Q32854).
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Illumina Casava1.8 software used for basecalling.
MeDIP-seq reads were aligned to the UCSC mm10 genome assembly using Rsubread or QuarR with the default configurations.
Duplicated reads were removed by Picard (http://broadinstitute.github.io/picard/)
peaks were called using Macs2 with the following setting:narrow peaks (P<1 ×10-6, FDR<0.01),
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files were generated from BAM files using deepTools bamCoverage with parameters –normalizeUsingRPKM –binsize 10
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| Submission date |
May 22, 2019 |
| Last update date |
May 23, 2019 |
| Contact name |
keren cheng |
| E-mail(s) |
keren.cheng@outlook.com
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| Organization name |
Zhejiang University
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| Department |
School of Medicine
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| Street address |
1575 chouzhou bei road
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| City |
Yiwu |
| State/province |
Zhejiang |
| ZIP/Postal code |
320000 |
| Country |
China |
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| Platform ID |
GPL21493 |
| Series (2) |
| GSE131654 |
Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis (MeDIP-seq) |
| GSE131657 |
Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis |
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| Relations |
| BioSample |
SAMN11832357 |
| SRA |
SRX5884228 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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