Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Unclassified
Antigen
Unclassified

Cell type

Cell type Class
Lung
Cell type
Lung
MeSH Description
Either of the pair of organs occupying the cavity of the thorax that effect the aeration of the blood.

Attributes by original data submitter

Sample

source_name
Lung
tissue
normal lung
genotype
EZH2 overexpressing

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Mouse lung tissue was pulverized using Covaris Tissue Smasher model CP02 by following the CryoPrep Dry Pulverization Manuel. Lung tissue was smashed 1-2 times on setting 4 in the tissueTUBE (Covaris #520071). Approximately 50mg of pulverized lung tissue was cross-linked with prewarmed 1% formaldehyde (ThermoScientific #28906 diluted in PBS) for 20 minutes at 37°C. The tissue was spun down at 1,000rpm for 2 minutes and quenched with 0.125M glycine in PBS + 0.5% BSA for 20 minutes at room temperature, spun down at 1,000rpm for 2 minutes and washed with PBS + 2x Protease Inhibitor Cocktail (PIC) (Roche #11873580001) + 5mM Sodium Butyrate (Millipore #19-137) then spun down at 1,000rpm for 2 minutes. The cross-linked tissue was then lysed with 390uL ChIP Lysis Buffer (1% SDS, 10nM EDTA pH8.0, 50mM Tris-HCl pH 8.0, 2x PIC and 5mM Sodium Butyrate) on ice for one hour. The lysate was split into 3 microTUBEs (Covaris #520045) and sheared on the Covaris E210 Series with 5% Duty Cycle, 5 Intensity, 200 Cycles per Burst for a total of 27 minutes. The sheared chromatin was spun down at 14,000rpm for 15 minutes at 4°C. An aliquot of input was saved while the remaining chromatin was snap frozen and stored at -80°C. Input was brought up to 100ul with TE, 10ug of RNAseA (Roche) added and incubated for 30 minutes at 37°C followed by addition of 100ug of Proteinase K (Roche) and incubation at 65°C overnight. Input was purified with Qiagen PCR Purification Kit (#28104) and quantified The prepared chromatin was thawed on ice while 10ug of antibodies against either H3K27ac (Abcam #Ab4729) or H3K27me3 (Cell Signaling #CS9733S) were conjugated to a mix of magnetic Protein A and Protein G coupled beads (Invitrogen #100.02D and #100.04D respectively) in the presence of 0.5% BSA in PBS with rotation at 4°C for 2 hours. Beads were washed 3 times with 0.5%BSA in PBS and either 5ug of chromatin was added to the H3K27ac ChIP or 10ug of chromatin was added to the H3K27me3 ChIP and rotated overnight at 4°C. The beads were washed 2 times with Tris based RIPA buffer (0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 7.4, 1mM EDTA pH 8.0, 0.1% Sodium Deoxycholate), 2 times with 0.3M NaCl RIPA (0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 7.4, 1mM EDTA pH 8.0, 0.1% Sodium Deoxycholate, 0.3M NaCl), 2 times with LiCl Buffer (250mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1mM EDTA pH 8.0, 10mM Tris-HCl pH 8.0) and 2 times with TE buffer pH 7.6 (Fisher Scientific cat. no. BP2474-1). The beads were resuspended in 100uL of TE and RNAseA and PK digested/reverse crosslinked and purified as described in the chromatin prep.

Sequencing Platform

instrument_model
NextSeq 500

mm10

Number of total reads
36992866
Reads aligned (%)
99.0
Duplicates removed (%)
6.7
Number of peaks
19501 (qval < 1E-05)

mm9

Number of total reads
36992866
Reads aligned (%)
98.9
Duplicates removed (%)
6.8
Number of peaks
19500 (qval < 1E-05)

Base call quality data from DBCLS SRA