GSM4557163: ChIP-seq WT input; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
LMNA
Cell type
Cell type Class
Breast
Cell type
MDA-MB-231
Primary Tissue
Breast
Site of Extraction
Effusion, Pleural
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
MDA-MB-231 breast cancer cells
cell line
MDA-MB-231
tissue
mammary gland/breast
cell type
epithelial, breast cancer cell line
age
51 years adult
gender
female
genotype
wild type
chip antibody
lamin A antibody (Abcam, catalog# ab26300, lot# GR3222248-1)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were collected and fixed with 1% formaldehyde (Sigma) at room temperature for 3 min, followed by 125 mM glycine (Amresco) quenching. About 0.8 M fixed cells for each replicate were resuspended with 1 ml hypotonic buffer (20 mM Hepes pH 7.9, 10 mM KCl, 1 mM EDTA, 10% Glycerol, 0.2% NP-40) and incubated in ice for 20 min. After centrifugation at 3000 rpm for 5 min at 4°C, the supernatant was discarded and the nuclei pellets were resuspended with 30 μl nuclei lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0). After lysis at 4°C for 30 min, add 70 μl dilution buffer ((0.01% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 200 mM NaCl) to totally 100 μl with 0.3% SDS. The sample is transferred t 200 μl PCR tubes at this step. Chromatin was sheared into mainly 200-800 bp fragments with Q800R sonicator, at 80% power for 4 min, with 15 s ON and 30 s OFF. After sonication, the Triton X-100 in system was added to finally 1%. The chromatin was centrifuged at 20,000 g for 15 min at 4°C. The supernatant was saved to new PCR tubes and precleared with 8 μl protein A dynabeads (Invitrogen) at 4°C for 1 h, followed by adding lamin A antibody (ab26300, Abcam). About 1/50 of chromatin was taken out as the input. The chromatin-antibody mixture was incubated overnight at 4°C. Each 15 μl Protein A dynabeads blocked by 1% BSA/PBS were added into chromatin-antibody mixture and rotated together for 4 h at 4°C. Beads were washed with 150 μl low salt buffer for 3 min × 3 times, then for 1 min with 150 μl high salt buffer, and finally transferred into new tubes with 150 μl low salt buffer. Then ChIPmentation method was applied to add adaptors to ChIPed fragments and input fragments. After pheno-chloroform purification, the adaptor-ligated fragments were amplified by Q5 polymerase (NEB) to make libraries with Illumina Nextera index primers. Cycles were determined in advance. ChIPed groups were amplified for 10 cycles and input groups were amplified for 8 cycles. After size selection for 200-1000 bp fragments, the libraries were quantified with Qubit for concentrations and sequenced with paired-end 150 bp reads on Nova 6000 platform (Illumina).