Antigen

Antigen Class

Histone

Antigen

H3K27me3

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.

Sample

source_name

Control Epithelium

strain background

C57BL/6

genotype/variation

ShhCre;Ezh2fl/+

age

E16.5

tissue

lung

cell type

epithelium

chip antibody

H3K27me3 antibody

chip antibody vendor

Millipore

library adapter

AD007

sequencing depth

21978677

mapped reads

18808865

molecule subtype

Immunoprecipitated DNA

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Freshly sorted EpCAM+ cells from Ezh2-deficient and control embryonic lungs at day E16.5 were cross-linked in 1 ml of freshly prepared 1% formaldehyde for 10 min at room temperature (RT). Cross-linking was stopped by adding 100 μl of 1.25M Glycine and 5 min incubation at RT. Cells were collected by centrifugation at 1000 x g for 5 min at 4C, resuspended in 200 ul of hypotonic buffer from Chromatrap Standard Pro-A ChIP spin column sonication kit (Chromatrap) containing 1 μl of proteinase inhibitor cocktail (PIC) (Sigma) and incubated on ice for 10 min. At this stage concentration of nuclei in the hypotonic buffer was estimated using a haemocytometer. Nuclei were collected by centrifugation at 5000 x g for 5 min at 4°C. Nuclear pellet was snap-frozen in dry ice and stored at -80°C. Nuclear pellets from several preparations were pooled together to a minimum of 400000-500000 cells per sample (2 samples per genotype) in 130 ul of cell lysis buffer (Chromatrap) containing 1 μl of PIC and incubated on ice for at least 10 min. The entire volume of lysate was transferred to a microTUBE (Covaris) and sonicated on Covaris S220 machine for 15 min under the following conditions: Duty Cycle 2%; Peak Incident Power 105 W; Cycles per Burst 200. Sonicated lysate was centrifuged at 10000 x g for 5 min at 4°C and resulting supernatant was collected and stored at -80°C. A 25 μl aliquot of lysate was combined with 5 μl of 1M NaHCO3, 5 μl of 5M NaCl, 15 μl dH20 and 1 μl of 20 mg/ml Proteinase K (Roche). The mix was incubated at 65°C for 2 hours and Proteinase K was inactivated by heating the mix to 95°C for 10 min. DNA was isolated by Phenol-Chloroform extraction followed by DNA precipitation and resuspended in 30 ul of nuclease-free H2O. The fragment size and DNA concentration were determined using D1K Screentape on Tapestation instrument (Agilent). 50 μl of sonicated lysate was combined with 450 μl Chromatin Dilution Buffer (Millipore), 10 μg H3K27me3 antibody (Millipore #07-449), 2.25 μl PIC (Sigma) and 40 ul of Magna ChIP protein A magnetic beads (Millipore #16-661). The chromatin slurry was incubated overnight with rotation at 4°C and beads were washed according to Magna ChIP A kit instructions (Millipore #17-610). Chromatin was eluted from the beads by resuspension in 100 μl elution buffer (1% SDS, 0.1M NaHCO3) containing 1 μl of 20 mg/ml Proteinase K (Roche) followed by incubation at 62°C for 2 h with shaking and Proteinase K inactivation at 95°C for 10 min. 2 IP reactions were set up for each sample and eluted chromatin corresponding to the same sample was combined at this stage. DNA was isolated by phenol-chloroform extraction followed by precipitation and resuspended in 30 μl of nuclease free water. 20-30 ng of immunoprecipitated DNA from each of the samples as well as 100 ng of whole genome extract were subjected to NGS library preparation using TruSeq Nano DNA Sample Preparation Kit (Illumina) following kit instructions with the following adjustments: fragmentation and size selection steps were omitted and 10 cycles of amplification were carried out during the fragment enrichment step. Resulting libraries were size selected using Pippin Prep DNA Size Selection System (Sage Science) to ensure fragment size below 900 bp. Libraries were pooled at equimolar concentrations and sequenced on HiSeq 2500 TruSeq SBS Kit v3 - HS reagents (Illumina) as 100 bp single end reads at AGRF.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

21978677

Reads aligned (%)

94.9

Duplicates removed (%)

18.0

Number of peaks

447 (qval < 1E-05)

mm9

Number of total reads

21978677

Reads aligned (%)

94.7

Duplicates removed (%)

18.1

Number of peaks

399 (qval < 1E-05)