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| Status |
Public on Jan 12, 2021 |
| Title |
Nanog_ChIP_in_WT _rep2 |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells (mESCs)
genotype/variation: wild type
chip antibody: Nanog (Cell Signaling Technology, Cat# 8822)
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| Treatment protocol |
Bach1-Flag expression was induced by treating DoxBach1 mESCs with 0.5 μg/μL doxycycline (Selleck chemicals, Houston, TX) for 6 hours.
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| Growth protocol |
mESCs (E14Tg2A line) were kindly provided by Dr. Fei Lan. mESCs were maintained in 90% Dulbecco’s Modified Eagle Medium (DMEM) (Life technologies, Carlsbad), 10% fetal bovine serum (FBS) (Life technologies, Carlsbad, CA), 1% non-essential amino acid (NEAA), 1% penicillin and streptomycin (100× stock) (Gibco-Thermo Fisher, Waltham, MA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and 200 Units/mL murine leukemia inhibitory factor (LIF) (ESGRO; Merck-Millipore, Germany). mESC expansion was performed in gelatin-coated flasks (BBI Life Sciences, China).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP) sequencing: Cells were cross-linked with 1% formaldehyde for 10 minutes, quenched with 0.125 M glycine for 10 minutes; then, the cell pellets were resuspended in high salt Lysis buffer (50 mM HEPES/KOH, PH 7.5, 500 mM NaCl, 1% Trixon-X 100, 0.05% SDS, 10 mM EDTA, and proteinase inhibitors), and sonicated with a Bioruptor (Diagenode) for 20 minutes in 30-second intervals to shear the chromatin into 200- to 400-bp lengths. Chromatin was immunoprecipitated with the indicated antibodies and Dynabeads protein A and protein G (Thermo Fisher, Waltham, MA). For, Bach1-Flag ChIP, DoxBach1 mESCs were treated with 0.5 μg/μL doxycycline (Selleck chemicals, Houston, TX) for 6 hours. Then cells were cross-linked, quenched, and washed as described above. Cell nucleus were extracted before sonication. And the sonicated chromatin was incubated with anti-Flag affinity M2 agarose gel (Sigma-Aldrich, St. Louis, MO) overnight at 4℃. RNA-seq: Non-ribosomal RNA was isolated from 1 μg total RNA by using a TrueLib Poly (A) mRNA Magnetic Isolation Module (Excell Bio, Shanghai, China), and sequencing libraries were prepared with the TrueLib mRNA Library Prep Kit for Illumina (ExCell Bio, Shanghai, China).
ChIP DNA libraries and input DNA samples were prepared as directed by protocol of KAPA Hyper Prep Kit for Illumina (Kapa Biosystems, Wilmington, MA).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Description |
WT-NANOG_S5_L001
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| Data processing |
The FASTQ data were trimmed adaptor by trim_galore (bioinformatics.babraham.ac.uk)
ChIP-seq data mapped to mouse genome mm9 with Bowtie2 in pair-end mode.
ChIP-seq peaks were called by MACS1.4 and MACS2 .
RNA-seq data were mapped to mouse genome mm9 using STAR (020201) and assembled by StringTie (1.3.4d) with parameter -eB (did not consider novel isoforms) to get RPKM.
The RPKM value of genes was converted to Transcripts per million (TPM) to eliminate the statistical biases inherent in the RPKM measure.
Differentially expressed genes were generated by limma with fold change >1.5 and p <0.05.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-delimited text files for ChIP-seq peaks in sample_1 to sample_13. Tab-delimited text files for RNA-seq RPKM and TPM for sample_18 to sample_24. Excel file for differentially expressed genes for sample_18 to sample_24.
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| Submission date |
Dec 23, 2019 |
| Last update date |
Jan 13, 2021 |
| Contact name |
Siqing Wang |
| E-mail(s) |
wangs16@chop.edu
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| Organization name |
Children’s Hospital of Philadelphia
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| Department |
Department of Pediatrics, Division of Hematology
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| Lab |
Gerd Blobel's Lab
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| Street address |
3401 Civic Center Blvd
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| City |
Philadelphia |
| State/province |
PENNSYLVANIA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE142519 |
BACH1 Recruits NANOG and Histone H3 Lysine 4 Methyltransferase MLL/SET1 Complexes to Regulate Enhancer-promoter Activity and Maintains Pluripotency |
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| Relations |
| BioSample |
SAMN13671997 |
| SRA |
SRX7433228 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4231204_11_Nanog_ChIP_in_WT_rep2_macs2_peaks.narrowPeak.gz |
186.1 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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