Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Others
Cell type
Tumor
NA
NA

Attributes by original data submitter

Sample

source_name
H3.3K27M_Tumor
genotype
H3.3K27M
sample type
Tumor
chip antibody
None

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed with 1% formaldehyde (Sigma). Fixed cell preparations were washed, pelleted and stored at -80°C. Sonication of lysed nuclei (lysed in a buffer containing 1% SDS) was performed on a BioRuptor UCD-300 for 60 cycles, 10s on 20s off, centrifuged every 15 cycles, chilled by 4°C water cooler. Samples were checked for sonication efficiency using the criteria of 150-500bp by gel electrophoresis. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and before ChIP reaction ~5% of sonicated drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing (see below). ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit. 25ul Protein A beads were washed and then incubated with 6ug of H3K27ac antibody (Diagenode, C15410196), and 2 million cells of sonicated cell lysate combined with protease inhibitors for 10 hr, followed by 20 min wash cycle with provided wash buffers. Reverse cross linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail (Life Technologies) at 65°C for 30 min followed by 2ul Proteinase K (Thermo Fisher Scientific) at 65°C for 30 min. Samples were then purified with QIAGEN MiniElute PCR purification kit as per manufacturers' protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol. Library preparation was carried out using Kapa HTP Illumina library preparation reagents. Briefly, 25ul of ChIP sample was incubated with 45ul end repair mix at 20°C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50ul buffer enzyme mix for 30°C 30 min, followed by PEG/NaCl purification. Adaptor ligation: bead bound sample was incubated with 45ul buffer enzyme mix and 5ul of different TruSeq DNA adapters (Illumina) for each sample, for 20°C 15 min, followed by PEG/NaCl purification (twice). Library enrichment: 12 cycles of PCR amplification. Size selection was performed after PCR using a 0.6x/0.8x ratio of Ampure XP beads (double size selection) set to collect 250-450bp fragments.

Sequencing Platform

instrument_model
Illumina NovaSeq 6000

hg38

Number of total reads
51591970
Reads aligned (%)
92.6
Duplicates removed (%)
19.9
Number of peaks
1026 (qval < 1E-05)

hg19

Number of total reads
51591970
Reads aligned (%)
91.6
Duplicates removed (%)
20.3
Number of peaks
576 (qval < 1E-05)

Base call quality data from DBCLS SRA