Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
ZNF768

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

source_name
U2OS cells
chip antibody
ZNF768 mAb 7D6

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin immunoprecipitation for ChIP-seq: Cells were crosslinked using a formaldehyde containing solution (10mM NaCl, 0,1mM EDTA pH 8, 0,05mM EGTA pH 8, 5mM HEPES pH 7.8 and 1% formaldehyde) for 10 minutes at 20°C, the reaction was quenched by the addition of glycin to a final concentration of 250uM for 5 minutes. Crosslinked cells were collected and washed twice with PBS before snap freezing in liquid nitrogen and storage at -80°C until subsequent use. Prior to sonication, the crosslinked cells were resuspended in lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100, 1X protease inhibitor cocktail) at 4°C for 20 minutes. Nuclei were collected by centrifugation and washed in a second buffer (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 1X protease inhibitor cocktail) for 10 minutes at 4°C then collected by centrifugation and resuspended in the shearing buffer (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1X protease inhibitor cocktail). Sonication was carried out in a Bioruptor Pico ultrasounds water bath (Diagenode B01060001) for 30 cycles of 30 sec ON and 30 sec OFF pulses in 4°C water. Sonicated extracts were centrifuged at high speed in the presence of 0,1% of Triton X-100 and snap frozen in liquid nitrogen then stored at -80°C until subsequent use. Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0,5% BSA PBS overnight at 4°C. Pre-coated beads were then washed and incubated with the sonicated chromatin extracts, the ChIP was carried out overnight at 4°C on a rotating wheel. The equivalent of 10 × 107 cells sonicated extract was used for each ZNF ChIP experiment for both cell lines. After incubation, the beads were washed 7 times with Wash buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate, 1X protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10mM Tris pH 8 and 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatine was eluted by two sequential incubations with 100µl Elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) at 65°C for 15 minutes. The two eluates were pooled and incubated at 65°C for 12 hours to reverse-crosslink the chromatin followed by treatment with RNase A (0.2µg/mL) at 37°C for two hours and Proteinase K (0.2µg/mL) at 55°C for two hours. The DNA was isolated by phenol:chloroform: isoamylalcohol (25:24:1 pH 8) extranction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). At least 1ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400Bp prior to end-repair, 3'end adenylation and adapter ligation. Library fragments were then directly amplified by 10 cycles of PCR.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
44612069
Reads aligned (%)
96.9
Duplicates removed (%)
2.9
Number of peaks
5991 (qval < 1E-05)

hg38

Number of total reads
44612069
Reads aligned (%)
97.9
Duplicates removed (%)
2.4
Number of peaks
6571 (qval < 1E-05)

Base call quality data from DBCLS SRA