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Sample GSM6710845

Query DataSets for GSM6710845

Status

Public on Feb 27, 2024

Title

ChIPseq CEBPA WT 24 ChIP input Rep 2

Sample type

SRA

Source name

B-cells

Organism

Homo sapiens

Characteristics

cell type: B-cells
age: 48h
chip antibody: anti-GFP, clone 3E6 A-11120, ThermoFisher

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

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

NextSeq 2000

Description

ChIP-seq

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

Submission date

Nov 04, 2022

Last update date

Feb 27, 2024

Contact name

Alexandre P Magalhaes

E-mail(s)

magalhae@molgen.mpg.de

Organization name

Max Planck Institute for Molecular Genetics

Street address

Ihnestrasse 63-73