GSM3752712: Breast cancer cell line, cSrcKO, 9327 KO input DNA; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Breast
Cell type
Breast tumor
NA
NA
Attributes by original data submitter
Sample
source_name
Breast cancer cell line
strain
FVB/N
cell line
Breast cancer cell line
genotype
MMTV-NIC/c-Src LoxP/LoxP
passages
10-15
tissue
mammary gland
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
NIC cells in 15 cm plates were fixed with a 1% final concentration of formaldehyde for 5 min at room temperature and then lysed and sonicated. Three plates of cells were pooled for each sample (approximately 3x107 cells). Equal amounts of chromatin from 2 individual cell lines of each genotype were diluted in 2.5X ChIP dilution buffer (EDTA 2 mM, NaCl 100 mM, Tris 20 mM, Triton 0.5%) + 100 μL of PBS+0.5% BSA and 750 ng of D. melanogaster chromatin and 0.4 µg of a D. melanogaster H2Av-specific antibody (Active Motif) were added to each sample. 5 μg of anti-H3K27me3 antibody immobilized overnight at 4ºC on 20 μl of Magna ChIP Protein A+G magnetic beads (Millipore) were added to each sample and immunoprecipitation was performed overnight at 4ºC. Beads were washed 3 times for 3 min at 4ºC with 1 mL LiCl buffer (Tris 100mM, LiCl 500 mM, Na-deoxycholate 1%) then once with 1 mL TE buffer. DNA was eluted with 150 μL of elution buffer (0.1M NaHCO3, 0.1% SDS) overnight at 65ºC. Chromatin immunoprecipitated DNA was purified using a QIAquick PCR purification kit (Qiagen) and eluted in 35 μL of elution buffer. Libraries were constructed with the Illumina TruSeq ChIP Library Preparation Kit using Illumina's standard protocols.