Antigen

Antigen Class

Input control

Antigen

Input control

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

lung

strain

mixed background

genotype

Ptend/d mouse

age

7 months

tumors

no

chip antibody

none

tissue

lung

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

lungs were removed from the Ptend/d mice, snap frozen in liquid N2 and stored at -80°C until shipment on dry ice to ActiveMotif company. For fixation, lungs were cut into small pieces in PBS + 1% formaldehyde and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycin (final) and tissue pieces were treated with a TissueTearer. Chromatin was isolated by disrupting the cells with a Dounce homogenizer. Lysates were sonicated using a Misonix Sonicator 3000 equipped with a microtip in order to shear the DNA to an average length of 300-500 bp. Lysates were cleared by centrifugation and stored at -80 C. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNaseA, proteinase K and heat for de-crosslinking, followed by phenol/chloroform extraction and ethanol precipitation. Purified DNA was quantified on a NanoDrop spectrophotometer. For each ChIP reaction, 30 ug of chromatin was precleared with protein A agarose beads (Invitrogen). ChIP reactions were set up using precleared chromatin and antibodies and incubated overnight at 4 C. Protein A agarose beads were added and incubation at 4 C was continued for another 3 hr. Immune complexes were washed two times each with a series of buffers consisting of the deoxycholate sonication buffer, high salt buffer, LiCl buffer, and TE buffer. Immune complexes were eluted from the beads with SDS buffer, and subjected to RNase treatment and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP and Input DNAs were prepared for amplification by converting overhangs into phosphorylated blunt ends and adding an adenine to the 3’-ends. Illumina genomic adapters were ligated and the sample was size-fractionated (200-250 bp) on a 2% agarose gel. After a final PCR amplification step (18 cycles), the resulting DNA libraries were quantified and sequenced on HiSeq 2000.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

42136034

Reads aligned (%)

98.8

Duplicates removed (%)

6.9

Number of peaks

571 (qval < 1E-05)

mm9

Number of total reads

42136034

Reads aligned (%)

98.7

Duplicates removed (%)

7.0

Number of peaks

618 (qval < 1E-05)