GSM4445811: DIPGXIII-KO1 SUZ12; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
SUZ12
Cell type
Cell type Class
Neural
Cell type
SU-DIPG-XIII
NA
NA
Attributes by original data submitter
Sample
source_name
brain tumor derived cell line & S2 cells (spike in)
cell line
SU-DIPGXIII
chip antibody
SUZ12 (Cell Signaling Technology, #3737)
genotype
H3.3 (H3F3A) K27M-KO by introducing frameshift indel to the H3F3A allele carrying K27M mutation and leaving wild-type allele
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 2% of sonicated drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing. 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 (Invitrogen) or 70ul of sheep anti-mouse IgG beads (Invitrogen) were washed and then incubated with antibodies (protein A beads with: anti-H3K27me3 (1:40, CST 9733), anti-H3K27me2 (1:50, CST 9728), anti-H3K36me2 (1:50, CST 2901), anti-H3K9me3 (1:66, Abcam ab8898); anti-mouse IgG beads with: anti-H3K27me1 (1:50, Active Motif 61015), anti-H3K36me3 (1:100, Active Motif 61021)), 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. ChIP reaction for SUZ12 was performed as follows: antibodies (anti-SUZ12 (1:150, CST 3737)) were conjugated by incubating with 40ul protein A beads at 4°C for 6 hours, then chromatin from ~4 million cells was added in RIPA buffer, incubated at 4°C o/n, washed using buffers from Ideal ChIP-seq Kit (1 wash with each buffer, corresponding to RIPA, RIPA+500mM NaCl, LiCl, TE), eluted from beads by incubating with Elution buffer for 30 minutes at room temperature. Reverse cross linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail at 65°C for 30 min followed by 2ul Proteinase K 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 or HyperPrep Illumina library preparation reagents, according to manufacturer's instructions.