GSM1545667: input Serum ChIPSeq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
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
Attributes by original data submitter
Sample
source_name
Colon cancer cell line
strain
HCT116
cell type
human colon cancer cell
antibody
none
serum
activated
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