Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Neural

Cell type

Glioblastoma

MeSH Description

A malignant form of astrocytoma histologically characterized by pleomorphism of cells, nuclear atypia, microhemorrhage, and necrosis. They may arise in any region of the central nervous system, with a predilection for the cerebral hemispheres, basal ganglia, and commissural pathways. Clinical presentation most frequently occurs in the fifth or sixth decade of life with focal neurologic signs or seizures.

Sample

source_name

glioblastoma xenograft tissue

tissue

GBM12-5199

tissue type

frozen xenograft tissue

cell cycle stage

asynchronization

tmz sensitivity

sensitive

chip antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Frozen tissues were cut, divided into 50 mg aliquots, and stored in tubes at -80°C. The frozen tissues were homogenized on ice for 15–30 seconds in 500 μL 1X PBS using a tissue grinder (ACTGene). Tissue homogenates or cultured cells were cross-linked with 1% formaldehyde (final concentration) for 10 min and quenched with 125 mM glycine for 5 min at room temperature and washed once with TBS. The pellets were resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. Lysates were divided into two aliquots and washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After re-suspending in 500 μL MNase digestion buffer containing a proteinase inhibitor cocktail (Sigma), the lysates were incubated in the presence of 1,000 units of MNase (NEB, Ipswich, MA, Cat.# M0247S) at 37 °C for 20 min with continuous mixing in thermal mixer (Fisher Scientific, Pittsburgh, PA). After adding the same volume of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 15 min (30 secs on / 30 secs off) using Bioruptor Twin (UCD-400) (Diagenode) and centrifuged at 21,130 x g for 10 min. The supernatants were collected and the chromatin content was estimated by the Qubit assay (Invitrogen). For the normalization of ChIP efficiency, S2 or Sf9 chromatin was added. The chromatin was then incubated with 5 µg of rabbit monoclonal anti-Ezh2 antibody (Cell Signaling, Cat.# 5246), 5 µg of rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling, Cat.# 9733), 2 µg of rabbit polyclonal anti-H3K27M antibody (Millipore, Cat.# abe419), or 5 µg of mouse monoclonal anti-Jarid2 antibody (Novus, Cat.# NB100-2214) on a rocker overnight. Protein G-magnetic beads (30 μl, Life Technologies) were added for 3 hours incubation. The beads were extensively washed with ChIP buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl buffer (10 mM Tris-HCl, pH8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65°C overnight. DNAs were purified using Min-Elute PCR purification kit (Qiagen) after the treatment of RNase A and proteinase K. ChIP-seq libraries were prepared from 10 ng ChIP and input DNA using the Ovation ultralow DR Multiplex kit (NuGEN). The ChIP-seq libraries were sequenced to 51 base pairs from both ends on an Illumina HiSeq 2000 instrument in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility. Briefly, for RNAseq, total RNA was extracted from cells. The RNA quality was further evaluated using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). The Illumina TruSeq RNA Sample preparation Kit v.4.1 (Illumina Inc., San Diego, CA) was used to prepare cDNA libraries from 2 µg of total RNA. Individual barcoded libraries were analyzed using Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing was carried out on an Illumina HiSeq 2000 machine (Illumina) at Mayo Clinic Medical Genomic Facility.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

hg38

Number of total reads

33546790

Reads aligned (%)

93.8

Duplicates removed (%)

0.9

Number of peaks

606 (qval < 1E-05)

hg19

Number of total reads

33546790

Reads aligned (%)

92.9

Duplicates removed (%)

1.7

Number of peaks

364 (qval < 1E-05)