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Status |
Public on May 23, 2019 |
Title |
ChIPseq.ID4eGFP_Dim_H3K27ac_Replicate1 |
Sample type |
SRA |
|
|
Source name |
Spermatogonia
|
Organism |
Mus musculus |
Characteristics |
strian: C57BL/6 tissue: testis age: P6 chip antibody: H3K27ac (Active Motif #39133)
|
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 |
FACS-sorted cells were pelleted and re-suspended in nuclear isolation buffer (Sigma #NUC101-1KT). Depending on input size, chromatin was fragmented for 5-7.5 min using MNase at 21°C or 37°C 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 |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 3000 |
|
|
Data processing |
Illumina Casava1.8 software used for basecalling. ChIP-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:broadpeaks,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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|
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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
|
Organization name |
Zhejiang University
|
Department |
School of Medicine
|
Street address |
1575 chouzhou bei road
|
City |
Yiwu |
State/province |
Zhejiang |
ZIP/Postal code |
320000 |
Country |
China |
|
|
Platform ID |
GPL21493 |
Series (2) |
GSE131656 |
Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis (ChIP-seq) |
GSE131657 |
Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis |
|
Relations |
BioSample |
SAMN11832402 |
SRA |
SRX5884266 |