Antigen

Antigen Class

TFs and others

Antigen

SFPQ

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)

Sample

source_name

U2OS

cell type

Bone osteosarcoma cells

cell line

U2OS

genome build

hg19

treatment

DMSO

antibody

SFPQ

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For ChIP, chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bp with a Branson 250 sonicator. Solubilized chromatin was immunoprecipitated with 2 μg antibodies against SFPQ, NONO, and TAF15 at 4 °C overnight. Antibody–chromatin complexes were pulled down with protein G Dynabeads, washed, and then eluted. After cross-link reversal and RNase A and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads. For TSA-UBF labelling, cells were fixed with paraformaldehyde and permeabilized. For TSA-SFPQ labelling, cells were pre-extracted and fixed with paraformaldehyde. Cells were incubated with UBF antibody (1:100) for 2 hr at 37 °C or SFPQ antibody (1:1,000) overnight at 4 °C, followed by incubation with mouse/rabbit-HRP secondary antibody for 1 hr at 37 °C. After washing, cells were incubated with 25 μM Tyramide-Biotin, 0.0015% H2O2, 50% Sucrose for 30 min at room temperature. After sequential wash, cells were lysed and incubated at 65 °C overnight to reverse the crosslink. After genomic DNA extraction and sonication, gDNA was subjected to pull-down by Dynabeads M-280 Streptavidin for 2 hr. The pull-down DNA was eluted from beads for 3 min at 95 °C and purified using QIAquick PCR Purification Kit. ChIP/TSA DNA samples were used to prepare sequencing libraries with Ultralow V2 DNA-Seq Library Preparation Kit

Sequencing Platform

instrument_model

NextSeq 550

hg38

Number of total reads

20942233

Reads aligned (%)

97.7

Duplicates removed (%)

12.1

Number of peaks

10696 (qval < 1E-05)

hg19

Number of total reads

20942233

Reads aligned (%)

96.8

Duplicates removed (%)

13.6

Number of peaks

10574 (qval < 1E-05)