Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K27me3

Cell type

Cell type Class
Spleen
Cell type
Spleen
MeSH Description
An encapsulated lymphatic organ through which venous blood filters.

Attributes by original data submitter

Sample

source_name
spleen, RBP-J mutant, H3K27me3 ChIP
strain/background
129Sv
genotype
RBP-J flox/flox;Ren1dcre/+
age
253 days
tumors
yes
tissue
spleen
chip antibody
H3K27me3 (Millipore Cat.#07-449 Lot. JBC1854858)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Spleens were removed from the mice, snap frozen in liquid N2 and stored at -80°C until shipment on dry ice to ActiveMotif. For fixation, spleens 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 (250-350 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
39908973
Reads aligned (%)
96.3
Duplicates removed (%)
15.8
Number of peaks
4848 (qval < 1E-05)

mm9

Number of total reads
39908973
Reads aligned (%)
96.3
Duplicates removed (%)
15.8
Number of peaks
4841 (qval < 1E-05)

Base call quality data from DBCLS SRA