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| Status |
Public on Dec 31, 2022 |
| Title |
ChIP_ESC_Ola_Stat2_CST_IFNb_0h_Rep5 |
| Sample type |
SRA |
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| Source name |
ChIP_ESC_Ola_Stat2_CST_IFNb_0h
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| Organism |
Mus musculus |
| Characteristics |
strain: 129/Ola
cell type: Embryonic stem cells
treatment: IFNb_0h
chip antibody: #72604; CST; #lot 2
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| Treatment protocol |
All cells were treated with 500U/ml of self-made interferon beta (16.6U/µl) for 1 hour or 6 hours
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| Growth protocol |
Embryonic stem cells: Cultured on 0.1 % gelatinized tissue flasks in ESC media. Cells were incubated at 37 °C and 5 % CO2. Media was PowerStem ESPro1 (PAN-Biotech; P04-77510K) with self-made LIF (1:1000).
Mouse embryonic fibroblasts: Cells were incubated at 37 °C and 5 % CO2. Media was DMEM (Gibco, 11880-28), 10% FCS (PAN-Biotech; P30-3602), 1x L-Glutamine (PAN-Biotech, P04-80050), 1x PenStrep (PAN-Biotech, P06-07050).
Neural progenitor cells: ESCs were differentiated into NPCs based on the Bible-protocol Bibel et al. 2007. 4-5*10^6 cultured ESCs were split onto a T75 UltraLow-BindingPlates (Corning, 3814) in 15 ml StemPAN media (PAN-biotech, P08-50500). After 4 days 5µM retinoic acid (Sigma-Aldrich, R2625) was added. On day 8, cells were platted on plates coated with 1% Matrigel (Corning, 356230) in neuronal base medium (Gibco, 211103049), G5 supplements (Gibco, 17503012), NSC supplements (Gibco, 17502048). Experiments were performed five days after neuronal plating.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq: . For ESCs, the cell pellet was resuspended in 200 µl ATAC lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl2, 3 mM MgCl2, 0.1 % NP-40) and incubate for at room temperature for 2 min. 20 µl ATAC reaction buffer containing 10 µl 2x transposase buffer and 2.5 µl Tn5 enzyme (Illumina) was added and samples were incubated at 37 °C for 30 min. Samples were purified. For MEFs, the lysis buffer contained additionally 1 % digitonin.
ATAC-seq: Purified samples were directly amplifed, barcoded and purified.
ChIP-seq for histone marks in ESC: Cells were crosslinked by 1 % Formaldehyde for 10 min at room temperature and stopped with 125 mM Glycine for 5 min. Samples of around 40 million cells were treated with 40 U of MNase for 15 min at 37 °C. The samples were shared for 15 min (Burst 200; Cycle 20%; Intensity 8). Replicate 1 was shared for 30 min and no MNase treatment was performed. 4µg of antibody was used per sample and incubated for 2 hours at 4 °C. Protein G-beads were added over night. Multiple low and high salt washes were performed and the samples were eluted. Samples were reverse crosslinked and stored at -20°C.
ChIP-seq for histone marks in ESCs: Libraries were made using NEBNext Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs, E7645)
ChIP-seq for STATs in ESCs and MEFs: The isolation of the chromatin was done with Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003)
ChIP-seq for STATs in ESCs and MEFs: Libraries were made using NEBNext Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs, E7645)
RNA-seq: Total RNA was isolated using NucleoSpin RNA Plus Kit (Macherey-Nagel, 740984.250). Depletion of rRNA was done with Ribo-Zero rRNA Removal Kit (Illumina, 15066012) for ESCs and MEFs and with NEBNext® rRNA Depletion Kit (New England Biolabs, E6350).
RNA-seq: Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7760).
scATAC-seq: The experiment was performed based on the standard manufacturer’s protocol Chromium Single Cell ATAC Reagent Kits (PN-1000110).
scRNA-seq: The experiment was performed based on the standard manufacturer’s protocol of Chromium Single Cell 3’ Reagent Kits v2 (PN-120237).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
ATAC-seq bam files: Raw reads were trimmed by Trimmomatic (v0.36) and mapped using Bowtie2 (v2.3.3.1). The mapped reads were with bedtools (v2.27.1) sorted, shifted by +4/-5bp to correct for transposase intragtion bias, duplicates and reads in blacklisted reads removed. Reads with quality score below 30 were removed.
ATAC-seq bed files: The sorted and quality filtered bam files were used for peak calling by MACS2 (v2.1.2).
ATAC Norm counttables: Bam files were used to count reads around peaks of STAT1p701 and STAT2 by bedtools (v2.27.1). Reads were normalized by sequencing depth, fragment length and enrichment to control.
ChIP bed files: Raw reads were mapped with Bowtie (v1.2.2). The mapped reads were sorted, duplicates and reads in blacklisted regions removed with bedtools (v2.27.1).
ChIP Norm counttables: Bam files were used to count reads around peaks of STAT1p701 and STAT2 by bedtools (v2.27.1). Reads were normalized by sequencing depth, fragment length and enrichment to control. For histon marks H3 signal was the control. For STAT ChIPs IgG_Rb_CST.
RNA-seq raw counttable: Ribosomal RNAs were removed by SortMeRNA (v2.1). Raw reads were aligned with STAR (v2.5.3a) and Aligned.sorted.ByCoord.out.bam and Aligned.to.Transcriptome.out.bam were created. STAR counted reads per genes in ReadsPerGene.out.tab files. These files were combined and the resulting raw count-table was the input for DESeq2.
RNA-seq count table TPM and FPKM: Ribosomal RNAs were removed by SortMeRNA (v2.1). Raw reads were aligned with STAR (v2.5.3a) and Aligned.sorted.ByCoord.out.bam and Aligned.to.Transcriptome.out.bam were created. The Aligned.to.Transcriptome.out.bam files from STAR were used as input for RSEM (v1.3.0) to create TPM and FPKM values.
RNA-seq DESeq result tables: Result tables for DESeq2 (1.24.0) for the comparisons of 1h IFNb with 0h; 6h IFNb with 0h and 6h with 1h IFNb.
scATAC-seq: Raw reads were applied to CellRanger (v1.1.0), a full automatic pipeline especially for scATAC-seq data done by the 10x Chromium.
scATAC-seq: The default output counttable for downstream analysis.
scRNA-seq: Raw reads were applied to CellRanger (v1.3.1), a full automatic pipeline especially for scRNA-seq data done by the 10x Chromium.
scRNA-seq counttable: The default output folders with the counttable for downstream analysis.
Genome_build: mm10
Supplementary_files_format_and_content: ATAC-seq bam files: Quality filtered mapped read files
Supplementary_files_format_and_content: ATAC-seq bed files: File containing coordinates of peaks and enrichment scores by MACS2
Supplementary_files_format_and_content: ChIP-seq bed files: File containing coordinates of peaks and enrichment scores by MACS2
Supplementary_files_format_and_content: ChIP-seq peak list for STAT1 and STAT2 by DiffBind: Peak lists for STAT1p701 and STAT2 replicates were anaylzed by DiffBind (v2.12.0) to create list of robust binding sites over replicates. Peaks at 1h and 6h of IFNb treatment were combined
Supplementary_files_format_and_content: RNA-seq raw counttable: Counttable containing ENSEMBL gene ID and the raw read counts for each gene over the replicates in ESCs, MEFs and NPCs
Supplementary_files_format_and_content: RNA-seq count table TPM and FPKM: Counttable with normalized TPM and FPKM values for each samples of ESCs, MEFs and NPCs. In addtition, the ENSEMBL gene ID, all transcript Ids and the annotated gene length.
Supplementary_files_format_and_content: RNA-seq DESeq result tables: Results table from DESeq2 of all genes for the three compariosns of 1h vs 0h, 6h vs 0h and 6h vs 1h. Each files contains the ENSEMBL gene ID, the normalized counts for all used samples, the base mean expression, the log2 fold change of the comaprison, the standard error of the log2 fold change, the Wald statistic, pvalue and adjusted pvalue.
Supplementary_files_format_and_content: scATAC: Counttable with a the coordinates of mapped fragments, the barcode of the fragment and the counts.
Supplementary_files_format_and_content: scRNA-seq: The folder contains the deault output of Cellranger. A list of detected barcodes in the sample. A List of identified genes in the sample as ENSEMBL ID and gene symbole. And a matix which is the counttable.
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| Submission date |
Nov 03, 2020 |
| Last update date |
Dec 31, 2022 |
| Contact name |
Markus Muckenhuber |
| E-mail(s) |
m.muckenhuber@dkfz.de
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| Organization name |
DKFZ
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| Lab |
AG Rippe
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| Street address |
Im Neuenheimer Feld 280
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| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE160764 |
Epigenetic signals that direct cell type specific interferon beta response in mouse cells |
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| Relations |
| BioSample |
SAMN16651139 |
| SRA |
SRX9426647 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4878823_ChIP_peaks_ESC_Ola_Stat2_CST_IFNb_0h_Rep5.bed.gz |
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(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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