Antigen

Antigen Class

TFs and others

Antigen

SUPT6H

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-SPT6-AID-C1

cell line

U2OS-SPT6-AID-C1

treatment

none

label

none

sirna

none

antibody

Novus Biologicals anti-SPT6, #NB100-2582;

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

ChIP-seq: Cells were treated with 1% formaldehyde for 5 min at 37°C. After cell lysis, nuclei were re-suspended in RIPA buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP40, 1% deoxycholic acid (DOC), 0.1% SDS, 1 mM EDTA) and DNA was fragmented to a size <300 bp using Covaris. Chromatin was immunoprecipitated with 15 µg of corresponding antibody for 6 hours. Chromatin was eluted with 1% SDS and crosslinking was reverted overnight. For purification, chloroform/phenol extraction was used. ChIP-seq: Libraries were prepared according to instructions accompanying the NEBNext Ultra™ II DNA Library Prep Kit for Illumina. Purified DNA was end-repaired, A-tailed, ligated to Illumina adaptors, size-selected (200 bp) and eluted. DNA fragments were amplified by 12 to 17 cycles of PCR and library size was tested with the Fragment Analyzer (Advanced Analytical). The amount of library DNA was quantified using a picogreen assay RNA-Seq: U2OS cell lysates were spiked 4sU-treated mouse T-cell lysates. Total RNA was isolated using QIAzol and purified with miRNeasy columns and biotinylated. This was followed by 4sU-RNA pulldown pulldown using streptavidin-MyOne beads, followed by purification with RNA minElute columns (Qiagen) RNA-Seq: 4sU-RNA was used for library preparation with NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and NEBNext Ultra II Directional RNA Library Prep kit. The libraries were amplified with 10-14 PCR cycles depending on the input RNA. The concentration and size distribution of the libraries were determined on a Fragment Analyzer using the NGS Fragment High Sensitivity Analysis Kit (1-6,000 bp; Agilent Technologies). The libraries were sequenced on the NextSeq500 Illumina platform for 75 cycles. Base calling was performed using Illumina's BaseSpace platform. 3' polyA mRNA-Seq: RNA was isolated using RLT buffer and purified with RNeasy columns and biotinylated. After addition of ERCC spike in (mix1) (Thermo Fisher Scientific), alkylation of RNA was carried out using 10 mM iodoacetamide (Thermo Fisher Scientific), and the reaction quenched with 1M DTT (Thermo Fisher Scientific). Alkylated RNA was purified with RNA minElute columns (Qiagen). 3' polyA mRNA-Seq: Library preparation was carried out with using QuantSeq kit (Lexogen), and fragments were amplified with 13 PCR cycles.

Sequencing Platform

instrument_model

NextSeq 500

hg38

Number of total reads

21068987

Reads aligned (%)

87.2

Duplicates removed (%)

4.8

Number of peaks

1976 (qval < 1E-05)

hg19

Number of total reads

21068987

Reads aligned (%)

86.9

Duplicates removed (%)

5.5

Number of peaks

1985 (qval < 1E-05)