Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H2AK119Ub

Cell type

Cell type Class
Breast
Cell type
MCF 10A
Primary Tissue
Breast
Tissue Diagnosis
Fibrocystic Disease

Attributes by original data submitter

Sample

source_name
MCF10A
cell line
MCF10A
cell type
Mammary gland fibrocystic disease epithelial cells
cell source
ATCC #CRL-10317
modifications
Parental cells
chip antibody
Cell Signaling # 8240

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were grown to 80% confluence on 150cm plates and fixed with 1% formaldehyde (Sigma-Aldrich cat# 252549) added directly to culture media for 15min shaking gently at RT. During crosslinking, 1.25M glycine solution (10X) was prepared, then added to plates at a final concentration of 0.125M for 5min shaking gently at RT. The supernatant was aspirated, cells washed 1x with PBS, and harvested on ice using cell scrapers into 15ml sonication tubes (Diagenode cat# C01020031). Cells were pelleted at 2000rpm for 3min at 4°C, washed 1x with 10ml cold PBS, and pelleted once more. Cell pellet was resuspended in 1.3ml cold ChIP Buffer (2 volumes of SDS ChIP Buffer [100mM NaCl, 50mM Tris-HCl pH 8.1, 5mM EDTA pH 8.0, 0.5% SDS] with 1 volume TXT ChIP Buffer [100mM Tris-HCl pH 8.6, 100mM NaCl, 5mM EDTA pH 8.0, 5% Triton X-100]). Cells were then sonicated on a Bioruptor Pico at 4°C for 20min of 30'' ON-OFF cycles. After sonication, samples were centrifuged at maximum speed for 15min at 4°C and supernatant transferred to a new 1.5ml microtube. To check sonication efficiency, 20µl of sonicated samples was transferred to a new microtube with 80µl PBS and incubated at 65°C for 3hr on an Eppendorf Thermomixer shaking at 1000rpm to decrosslink. DNA was purified with the QIAquick PCR Purification Kit (Qiagen cat# 28106) and eluted in 30µl H2O. 6µl of orange DNA dye was added and 12µl and 24µl of each sample were run in a 1% agarose gel at 100V for 15min and imaged on a Bio-rad ChemiDoc XRS+. Protein concentration in sonicated samples was measured by Bradford assay, and 200µg of total protein was transferred to a 1.5ml LoBind tube (Eppendorf cat# 0030108051) and brought up to 500µl final volume with ChIP buffer. 5µl was removed as input material (1%) and placed in a separate microtube at 4°C. 2µg antibody was used for each histone ChIP (see Key Resources table for list of antibodies). The samples were rotated end-to-end overnight at 4°C. Protein A sepharose beads (GE Healthcare cat# 17528001) were washed 3x with ChIP buffer and 30µl bead slurry was added to each sample. Samples were incubated with beads for 2hr at 4°C rotating end-to-end. Following incubation, samples were centrifuged at 2000rpm for 3min at 4°C, washed 2x with ChIP Low Salt Buffer (50mM HEPES pH7.5, 140mM NaCl, 1% Triton X-100, 1x protease inhibitors), and 1x with ChIP High Salt Buffer (50mM HEPES pH7.5, 500mM NaCl, 1% Triton X-100, 1x protease inhibitors). Beads were dried after the last wash with a 28G needle fitted to a 1ml syringe. Elution Buffer (1% SDS, 0.1M sodium carbonate) was prepared fresh before use and 110µl was added to each sample and 95µl to each input sample that was previously set aside. To elute immunocomplexes from beads, samples were incubated at 65°C for 3hr on an Eppendorf Thermomixer shaking at 1000rpm. Tubes were centrifuged for 3min at 2000rpm at RT and 100µl of supernatant was transferred to a new tube, being careful not to aspirate beads. DNA purification was performed with the QIAquick PCR Purification Kit (Qiagen cat# 28106) and eluted in 60µl H2O and quantified by Qubit. Immunoprecipitated DNA was used to generate libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs cat# 7370) following the manufacturer's instructions.

Sequencing Platform

instrument_model
NextSeq 500

hg38

Number of total reads
76601778
Reads aligned (%)
98.5
Duplicates removed (%)
16.2
Number of peaks
1135 (qval < 1E-05)

hg19

Number of total reads
76601778
Reads aligned (%)
98.0
Duplicates removed (%)
17.2
Number of peaks
986 (qval < 1E-05)

Base call quality data from DBCLS SRA