Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Neural

Cell type

Medulloblastoma

MeSH Description

A malignant neoplasm that may be classified either as a glioma or as a primitive neuroectodermal tumor of childhood (see NEUROECTODERMAL TUMOR, PRIMITIVE). The tumor occurs most frequently in the first decade of life with the most typical location being the cerebellar vermis. Histologic features include a high degree of cellularity, frequent mitotic figures, and a tendency for the cells to organize into sheets or form rosettes. Medulloblastoma have a high propensity to spread throughout the craniospinal intradural axis. (From DeVita et al., Cancer: Principles and Practice of Oncology, 5th ed, pp2060-1)

Sample

source_name

input DNA

developmental stage

P13

tissue

medulloblastoma

genotype

Sufu;Spop double knockout

chip antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Tumours were dissected, cut into small pieces, and crosslinked in solution A (1% formaldehyde, 50mM Hepes-KOH, 100 mM NaCl, 1mM EDTA, 0.5 mM EGTA) for 20 minutes at room temperature. 1/20 volume of 2.5 M glycine was added to quench formaldehyde. Tissues were rinsed in ice-cold PBS and dounced in Wheaton tissue grinder. Cell suspensions were passed through a 100μm cell strainer to remove connective tissues, then centrifuged at 4 °C at 2500 × rcf for 3 min, followed by a wash in PBS. Olig2 antibody (EMD Millipore, ab15328) was incubated with pre-blocked magnetic beads in 250μL total volume for 8 hours at 4 °C, then washed and resuspended in 100μL 0.5% BSA (w/v) solution. Cell pellets were lysed by sequentially resuspending and centrifuging in LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100), and LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA). Pellet was resuspended in LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine) and sonicated into 200-400bp fragments. Sonicated lysates were diluted with LB3. After saving 50μL for input control, they were split into tubes containing 10% v/v Triton X-100 with Gli2 (homemade), H3K27ac (Millipore 05-1334 lot 2489078), or H3K36me3 (Abcam ab9050 lot GR204353-1) antibody, and incubated overnight. Beads were washed 6 times in RIPA buffer (50 mM Hepes-KOH, pH 7.5; 500 mM LiCl; 1mM EDTA; 1% NP-40 or Igepal CA-630; 0.7% Na-Deoxycholate), and once in TBS (20 mM Tris-HCl, pH 7.6; 150 mM NaCl). Protein-DNA complexes were eluted and crosslink reversed with 200 μL of elution buffer (50 mM Tris-HCl, pH 8; 10 mM EDTA; 1% SDS). The 50μL input lysate was also included in this step. Eluted DNA was diluted with 200 μL of TE, and treated with RNaseA for 30 min at 37°C, and subsequently with proteinase K for 2 hours at 55°C. Phenol:chloroform:isoamyl alcohol was used to extract DNA. Libraries were prepared according to manfacturer's instructions for the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® kit.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

14501564

Reads aligned (%)

73.3

Duplicates removed (%)

4.7

Number of peaks

129 (qval < 1E-05)

mm9

Number of total reads

14501564

Reads aligned (%)

73.3

Duplicates removed (%)

4.6

Number of peaks

91 (qval < 1E-05)