GSM1280519: HMLE Parental H3K4me3; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K4me3
Cell type
Cell type Class
Breast
Cell type
HMLE
NA
NA
Attributes by original data submitter
Sample
source_name
HMLE_Parental_H3K4me3
cell type
human mammary epithelial cells
transfected with
none (parental)
culture type
monolayer
chip antibody
H3K4me3
chip antibody vendor
Millipore
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. For each chromatin immunoprecipitation (ChIP) reaction, 0.5 million cells were used. ChIP was performed using Dynabeads Protein G (Invitrogen, Carlsbad, CA) as per manufacturer’s protocol. ChIP libraries for sequencing were prepared as per Illumina’s standard protocol (Illumina, San Diego, CA) with minor modifications. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, Klenow enzyme (DNA Polymerase I Large Fragment) and T4 polynucleotide kinase (NEB, Ipswich, MA). The blunt phosphorylated DNA fragments were treated with Klenow fragment (3’ to 5’ exo minus) (NEB) and dATP to add an ‘A’ base to the 3’ end. Sequencing adapters were ligated to the ends of the DNA fragments using Quick T4 DNA Ligase (Enzymatics, Beverly, MA) and Genomic Adapter Oligo Mix (Illumina, part # 1000521). The ligation products were size selected by electrophoresis in a 2% agarose gel. A slice corresponding to 300 (± 25) bp size window based on DNA ladder was cut out and DNA is extracted from agarose. Eluted DNA was amplified by 14 cycles of PCR using Phusion High-Fidelity DNA Polymerase (NEB) and Illumina’s PCR primer 1.1 and PCR primer 2.1. Resulting sequencing library was cleaned with AMPure magnetic beads (Agencourt, Beverly, MA) and ready for single-read sequencing on Illumina Genome Analyzer II.