Antigen

Antigen Class

Input control

Antigen

Input control

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 cells

strain

ATCC HTB-96

tissue

bone

genotype

osteosacoma

chip antibody

IgG santa cruz sc-2027 X

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

total RNA was extracted using a RNeasy MinElute Cleanup Kit NEBNext DNA Library Prep Master Mix Set for Illumina for RNA-seq and NEXTflex ChIP-seq Kit, Bioo Scientific for chip-seq RNA-seq: total RNA was extracted using a RNeasy MinElute Cleanup Kit (QIAGEN, Catalog no. 74204). Double-stranded cDNA was synthesized according to the mRNA Sequencing Sample Preparation Guide (part#1004898 Rev.D, Illumina, San Diego, CA) and single-end libraries were prepared using the NEBNext DNA Library Prep Master Mix Set for Illumina (New England BioLabs, Catalog no.74204 E6040L). For RNA-seq of RNaseH treated MEF cells, the protocols were mainly adopted as previously described (Nat Methods 10: 623-629). For qRNA-seq of MEF cells, the GeneRead rRNA Depletion Kit (Qiagen) was used to remove ribosome RNA. Nascent RNA-seq of U2OS cells was performed as previously described (Science 322: 1845-1848), with the exception of not applying a strand specific method. ChIP-seq procedure as previously described (Cell. 2013 May 9;153(4):855-68). For the Bmal1 and IgG ChIP-seq, the RIPA buffer was diluted 1 to 10. For HIF1A CHIP-seq, protocols are used as described (ChIP-IT high sensitive kit (Cat# 53040, Active Motif)). The library was constructed using a commercially available kit (NEXTflex ChIP-seq Kit, Bioo Scientific). Samples were indexed using NEXTflex™ DNA Barcodes (Bioo Scientific). The library was qualified using an Agilent 2100 Bioanalyzer (Agilent) and quantified using KAPA Library Quantification Kits (Kappa Biosystems). Finally, the indexed libraries were sequenced using a HiSeq 2500 (Illumina).

Sequencing Platform

instrument_model

Illumina HiSeq 2500

hg38

Number of total reads

30358620

Reads aligned (%)

48.5

Duplicates removed (%)

66.6

Number of peaks

234 (qval < 1E-05)

hg19

Number of total reads

30358620

Reads aligned (%)

48.2

Duplicates removed (%)

68.3

Number of peaks

277 (qval < 1E-05)