|
| Status |
Public on May 23, 2019 |
| Title |
ChIPseq.ID4eGFP_Dim_H3K27me3_Replicate3 |
| Sample type |
SRA |
| |
|
| Source name |
Spermatogonia
|
| Organism |
Mus musculus |
| Characteristics |
strian: C57BL/6
tissue: testis
age: P6
chip antibody: H3K27me3 ( Abcam #ab6002)
|
| 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).
|
| |
|
| 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
|
| |
|
| 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 |
SAMN11832394 |
| SRA |
SRX5884274 |
