Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
TP53BP1

Cell type

Cell type Class
Bone
Cell type
U2OS
Tissue
bone
Lineage
mesoderm
Description
osteosarcoma from the tibia of a 15 year old, J. Ponten and E. Saksela derived this line (originally 2T) in 1964 from a moderately differentiated sarcoma, viruses were not detected during co-cultivation with WI-38 cells or in CF tests against SV40, RSV or adenoviruses, mycoplasma contamination was detected and eliminated in 1972, (PMID: 6081590)

Attributes by original data submitter

Sample

Alias
E-MTAB-58171533046345:53BP1_AS_pOHT
Broker name
ArrayExpress
Description
Protocols: DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma; H7904) for 4h For ChIP:Formaldehyde was added to the culture medium at a final concentration of 1% and crosslinking was allowed to proceed for 15 min at room temperature. Reaction was stopped by adding glycine (0.125M final concentration) and incubation for 5 minutes at room temperature. Cells were washed twice with PBS and harvested by scraping. Cell lysis was performed by incubation in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% IGEPAL® CA-630) followed by Dounce homogenization. Nuclei were harvested by centrifugation and incubated in nuclear lysis buffer: (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated 10 times for 10 s at a power setting of 5 and 50% duty cycle (Branson Sonifier 250). Samples were diluted 10 times in ChIP dilution buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X‐100, 0.01% SDS) and precleared using a mixture of protein A-agarose (Pierce) and protein G-sepharose (Sigma) previously blocked with BSA (Sigma). Precleared samples (10 to 200 μg) were incubated overnight at 4°C with indicated antibodies. Immune complexes were recovered by incubation with blocked protein A/protein G beads for 2 h at 4°C on a rotating wheel. Beads were washed once in dialysis buffer (50 mM Tris pH 8, 2 mM EDTA, 0.2% Sarkosyl), five times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1% IGEPAL® CA-630, 1% NaDoc) and twice with TE (10mM Tris-HCl, pH 8, 1mM EDTA). Beads were resuspended in TE supplemented with 50µg/ml DNase-free RNase A (Abcam) and incubated for 30 minutes at 37°C. SDS (final concentration 0.5%) was added and crosslink was reversed by overnight incubation at 70°C. Following a 2 hours treatment with 200 µg/ml proteinase K (Roche) at 45°C, DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation in presence of 5 µg glycogen (Invitrogen). DNA pellet was resuspended in water. Prior to next-generation sequencing library preparation, samples from multiples ChIP experiments were pooled and sonicated for 5 cycles (30 seconds on, 30 seconds off, high setting) on a Bioruptor (Diagenode) then concentrated with a vacuum concentrator (Eppendorf). Libraries for H3K4me2, H4K20me1, H3K36me3, H3K79me2, H2Bub, H3K56ac, H4K16ac, H3K9me2, H4S1P, H3K36me2 and γH2AX were prepared BGI (Beijin Genomics Institute, Hong Kong) using proprietary protocols. Libraries for FK2 samples were prepared at GATC biotech (Konstanz, Germany) using proprietary protocols. Libraries for H2BK120ac, H2AZ, H1, MacroH2A, H4K20me3, 53BP1, H4K12ac, H2AZac and H3 were prepared atEMBL Genomics core facilities (Heidelberg, Germany) using NEBNext ChIP-Seq Library Prep kit (New England Biolabs). Libraries for BLESS were prepared using TruSeq Nano DNA LT Library Preparation Kit (Illumina).
ENA checklist
ERC000011
INSDC center alias
Universite de Toulouse ; UPS ; LBCMCP ; 118 route de Narbonne, 31062, Toulouse, France;CNRS ; LBCMCP ; F-31062 Toulouse, France
INSDC center name
Universite de Toulouse ; UPS ; LBCMCP ; 118 route de Narbonne, 31062, Toulouse, France;CNRS ; LBCMCP ; F-31062 Toulouse, France
INSDC first public
2018-09-28T13:00:54Z
INSDC last update
2018-07-31T15:21:41Z
INSDC status
public
SRA accession
ERS2630358
Sample Name
ERS2630358
Title
53BP1_AS_pOHT
cell_line
U2OS
genotype
AsiSI-ER expression
organism
Homo sapiens
sex
female

Sequenced DNA Library

library_name
53BP1_AS_pOHT_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma; H7904) for 4h For ChIP:Formaldehyde was added to the culture medium at a final concentration of 1% and crosslinking was allowed to proceed for 15 min at room temperature. Reaction was stopped by adding glycine (0.125M final concentration) and incubation for 5 minutes at room temperature. Cells were washed twice with PBS and harvested by scraping. Cell lysis was performed by incubation in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% IGEPAL® CA-630) followed by Dounce homogenization. Nuclei were harvested by centrifugation and incubated in nuclear lysis buffer: (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated 10 times for 10 s at a power setting of 5 and 50% duty cycle (Branson Sonifier 250). Samples were diluted 10 times in ChIP dilution buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X‐100, 0.01% SDS) and precleared using a mixture of protein A-agarose (Pierce) and protein G-sepharose (Sigma) previously blocked with BSA (Sigma). Precleared samples (10 to 200 μg) were incubated overnight at 4°C with indicated antibodies. Immune complexes were recovered by incubation with blocked protein A/protein G beads for 2 h at 4°C on a rotating wheel. Beads were washed once in dialysis buffer (50 mM Tris pH 8, 2 mM EDTA, 0.2% Sarkosyl), five times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1% IGEPAL® CA-630, 1% NaDoc) and twice with TE (10mM Tris-HCl, pH 8, 1mM EDTA). Beads were resuspended in TE supplemented with 50µg/ml DNase-free RNase A (Abcam) and incubated for 30 minutes at 37°C. SDS (final concentration 0.5%) was added and crosslink was reversed by overnight incubation at 70°C. Following a 2 hours treatment with 200 µg/ml proteinase K (Roche) at 45°C, DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation in presence of 5 µg glycogen (Invitrogen). DNA pellet was resuspended in water. Prior to next-generation sequencing library preparation, samples from multiples ChIP experiments were pooled and sonicated for 5 cycles (30 seconds on, 30 seconds off, high setting) on a Bioruptor (Diagenode) then concentrated with a vacuum concentrator (Eppendorf). Libraries for H3K4me2, H4K20me1, H3K36me3, H3K79me2, H2Bub, H3K56ac, H4K16ac, H3K9me2, H4S1P, H3K36me2 and γH2AX were prepared BGI (Beijin Genomics Institute, Hong Kong) using proprietary protocols. Libraries for FK2 samples were prepared at GATC biotech (Konstanz, Germany) using proprietary protocols. Libraries for H2BK120ac, H2AZ, H1, MacroH2A, H4K20me3, 53BP1, H4K12ac, H2AZac and H3 were prepared atEMBL Genomics core facilities (Heidelberg, Germany) using NEBNext ChIP-Seq Library Prep kit (New England Biolabs). Libraries for BLESS were prepared using TruSeq Nano DNA LT Library Preparation Kit (Illumina).

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
129041155
Reads aligned (%)
95.2
Duplicates removed (%)
26.7
Number of peaks
1379 (qval < 1E-05)

hg38

Number of total reads
129041155
Reads aligned (%)
95.7
Duplicates removed (%)
25.4
Number of peaks
2224 (qval < 1E-05)

Base call quality data from DBCLS SRA