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| Status |
Public on Nov 22, 2022 |
| Title |
EBF1-dTAG_C, 0h, input |
| Sample type |
SRA |
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| Source name |
A-MuLV transformed pro-B cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: A-MuLV transformed pro-B cells
genotype: EBF1-dTAG_C
treatment: DMSO 6h
chip antibody: none
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| Treatment protocol |
Cells were treated with dTAG-13 (final concentration 0.5 uM) or DMSO for 6 hours.
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| Growth protocol |
Production of A-MuLV transformed cells with the replacement of endogenous EBF1 by exogenous modified genes was described in Boller et al. 2016. A-MuLV transformed cells were cultured in the RPMI 1640 medium with 10% FCS; 1x Penicillin-Streptomycin-Glutamine; 0.06 mM β-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
EBF1 ChIP was performed according to Boller et al. 2016 protocol with minor changes. Cells were crosslinked with formaldehyde. 4 µg rabbit a-EBF1 (1C) rabbit antibody per sample was used. Cells were harvested and washed with PBS. Cell pellet was resuspended in PBS with 2% FCS (4 mln cells per 1 ml buffer). Fresh formaldehyde mix (50mM HEPES-KOH pH8.0, 100mM NaCl, 0.5mM EGTA, 1mM EDTA, 11% (v/v) formaldehyde) was added to the cell suspension to a final concentration of 1% FA. Tubes were incubated on rotator for 10 min at RT. Quenching was performed by adding 2 M Glycine to the final concentration 0.2 M. Cell pellet was resuspended in Lysis buffer (50mM Tris-HCl pH 8.0, 1% (w/v) SDS, 10mM EDTA; PIM) (4 mln cells per 100 μl buffer). Chromatin was sheared the chromatin with a Bioruptor (20-25 cycles, output level "High", 30 sec ON, 30 sec OFF, 4°C). Fragment size and amount of chromatin was checked. 10-20 μg DNA was used for 1 ChIP reaction. Chromatin was mixed with 4 μg of EBF1 antibody or rabbit IgG and incubated on rotator ON at 4°C. Chromatin was incubated with 30 μl of Dynabeads Protein G (ThermoFisherScientific) suspension per sample on rotator for 4 hours at 4°C. Beads were washed 4 times with Wash buffer A (20mM Tris-HCl pH 8.0, 150mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2mM EDTA); once with Final wash buffer A (20mM Tris-HCl pH 8.0, 500mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2mM EDTA). Every wash was on rotator for 5 minutes at 4°C. Chromatin was eluted with 100 μl Elution buffer (10 mM Tris-HCl, pH 8.0; 0.5% SDS; 300 mM NaCl; 5 mM EDTA pH 8.0) by shaking vigorously at 65°C for 30 min. Samples were revese crosslinked, treated with RNase A and Proteinase K, and purified with QIAquick PCR Purification Kit.
NEBNext Ultra II DNA Library Prep Kit for Illumina was used for library preparation according to the manufacturer's protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Quality control and read mapping was done using snakePipes (v2.5.1), an automatic pipeline for analysis of deep sequencing data.
Reads were mapped to the mouse genome (GRCm38 – mm10) using bowtie2 (v2.3.5.1).
Reads were sorted and indexed using samtools (v1.10) and duplicates were removed by sambamba_markdup.
Coverage bigwig files were created using bamCoverage from deeptools (v3.3.2) with normalization on sequencing depth (CPM).
Peak calling was performed using MACS2 (v2.2.7.1) with default settings.
Assembly: mm10
Supplementary files format and content: One .narrowPeak file containing the MACS2 output for each ChIP sample.
Supplementary files format and content: One bigwig file (.bw) for each sample.
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| Submission date |
Apr 20, 2022 |
| Last update date |
Nov 22, 2022 |
| Contact name |
Nikolay Zolotarev |
| E-mail(s) |
zolotarev@ie-freiburg.mpg.de
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| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
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| Department |
Cellular and Molecular Immunology
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| Lab |
Laboratory Rudolf Grosschedl
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| Street address |
Stübeweg, 51
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| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
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| Relations |
| BioSample |
SAMN27671605 |
| SRA |
SRX14935390 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6051629_EBF1-dTAG_C_0h_input.bw |
164.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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