Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Digestive tract

Cell type

HCT 116

Primary Tissue

Colon

Tissue Diagnosis

Carcinoma

Sample

source_name

Colon cancer cell line

strain

HCT116

cell type

human colon cancer cell

antibody

none

serum

starved

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For ChIP-seq, HCT116 cells were crosslinked with 1% paraformaldehyde for 10 minutes at room temperature with gentle rotation, and then quenched by 0.125 M glycine solution. After washing, nuclei were sonicated on a Misonix Sonicator 3000 Ultrasonic Cell Disruptor, and the supernatant was used for immunoprecipitation with the indicated antibody. ChIP-sequencing libraries were prepared with Illumina’s Tru-seq DNA sample prep kit. For nascent RNA-seq, HCT116 cells was harvested and washed 3 times with cold PBS, then suspended in 10 ml Buffer A (10 mM HEPES at pH=7.9, 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 1× Complete protease inhibitors [Roche]). After incubating on ice for 15 minutes, the cells were homogenized in pre-cooled 15 ml Dounce tissue homogenizer for 15 times. Nuclei were then washed twice with Buffer B (10 mM HEPES at pH=7.9, 250 mM Sucrose, 1 mM DTT, 1× Complete protease inhibitors). The pellet was vigorously suspended with 1 ml NUN buffer (20 mM HEPES at pH=7.9, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% v/v Nonidet P40, 1 mM DTT, 20 U/ml SUPERase.In RNase Inhibitor [Ambion]) that was freshly prepared. The chromatin was then washed twice more with 5 ml NUN buffer each time. The supernatant was removed and TRIzol reagent (Invitrogen) was added to the pellet for purification of RNA. The RNA was subjected to polyA depletion with Oligo(dT) magnetic beads (Invitrogen) and DNase I treatment (NEB) for 20 minutes, and then was re-purified. For sequencing, 2 μg of resulting RNA was used for ribosomal RNA depletion with the RiboZero kit (Epicenter) and libraries were made with the TruSeq RNA sample Prep Kit (Illumina). ChIP-seq and Nascent-RNA-seq libraries were prepared for sequencing using standard Illumina protocols

Sequencing Platform

instrument_model

Illumina HiSeq 2000

hg38

Number of total reads

32157370

Reads aligned (%)

98.9

Duplicates removed (%)

4.8

Number of peaks

971 (qval < 1E-05)

hg19

Number of total reads

32157370

Reads aligned (%)

97.8

Duplicates removed (%)

6.0

Number of peaks

1109 (qval < 1E-05)