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Sample GSM3791849 Query DataSets for GSM3791849
Status Public on May 23, 2019
Title MeDIPseq.ID4eGFP_Bright_Replicate3
Sample type SRA
 
Source name Spermatogonia
Organism Mus musculus
Characteristics strian: C57BL/6
tissue: testis
age: P6
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.
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).
 
Library strategy MeDIP-Seq
Library source genomic
Library selection 5-methylcytidine antibody
Instrument model Illumina HiSeq 3000
 
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
 
Submission date May 22, 2019
Last update date May 23, 2019
Contact name keren cheng
E-mail(s) keren.cheng@outlook.com
Organization name University of Pennsylvania
Department Biomedical Sciences
Street address 3800 Spruce Street
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
 
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
Relations
BioSample SAMN11832358
SRA SRX5884227

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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