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Status |
Public on Feb 27, 2024 |
Title |
ChIPseq CEBPA WT 48 ChIP Rep 1 |
Sample type |
SRA |
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Source name |
B-cells
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Organism |
Homo sapiens |
Characteristics |
cell type: B-cells age: 48h chip antibody: anti-GFP, clone 3E6 A-11120, ThermoFisher
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Extracted molecule |
genomic DNA |
Extraction protocol |
Five million cells were collected and cross-linked for 10 min using 1% formaldehyde and quenched using a final concentration of 0.125 M glycine. After a wash in cold PBS and centrifugation, pellets were lysed in 500 µl pre-cooled SDS lysis buffer (1% SDS, 10mM EDTA, 50 mM Tris pH8 and 1x PIC) and incubated on ice for 15 min. Chromatin was sheared on a Bioruptor pico sonicator (Diagenode) at 4°C for 18 cycles of 30s ON and 30s OFF. After sonication, the solution was clarified by centrifugation at 1,000g for 5 min at 4°C and supernatant was transferred to a low-bind tube and mixed with 900µl of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1X PIC) containing antibody-coupled beads (10µL anti-GFP, clone 3E6 A-11120, ThermoFisher and 35 µL of protein G magnetic beads, 10003D, ThermoFisher). Five percent were saved as input and the samples were incubated overnight at 4°C under rotation. The beads were then collected and washed with 500µL of low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), RIPA-LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-DOC) and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were then collected and eluted in 70µL of Elution buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 300 mM NaCl, 0.5% SDS) and incubated with proteinase K for 1h at 55°C and then overnight at 65°C to reverse the cross-linking. Beads were collected and transferred to a new tube and a second step of elution was performed with 30 µL of Elution buffer. DNA was finally purified with a Qiagen MinElute column 3 ng of DNA were used to construct sequencing libraries using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (E7370L). Single-end 50bp with 50 million reads
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
NextSeq 2000 |
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Description |
ChIP-seq
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Data processing |
Reads were mapped to genome using BWA and samtools was used for SAM to BAM conversion, sorting and indexing. Genome Analysis Toolkit v4 was used to remove duplicated reads. Peak calling was then performed using MACS3 v3.0.0 b1 using the input of the respective sample genome_build: hg38
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Submission date |
Nov 04, 2022 |
Last update date |
Feb 27, 2024 |
Contact name |
Alexandre P Magalhaes |
E-mail(s) |
magalhae@molgen.mpg.de
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Organization name |
Max Planck Institute for Molecular Genetics
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Street address |
Ihnestrasse 63-73
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City |
Berlin |
ZIP/Postal code |
14194 |
Country |
Germany |
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Platform ID |
GPL30173 |
Series (1) |
GSE201655 |
An activity-specificity trade-off encoded in human transcription factors |
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Relations |
BioSample |
SAMN31610403 |
SRA |
SRX18173773 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6710838_WT_48_ChIP_rep1_peaks.narrowPeak.gz |
752.1 Kb |
(ftp)(http) |
NARROWPEAK |
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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