GSM1388026: 445a input-DNA; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Unclassified
Cell type
Unclassified
NA
NA
Attributes by original data submitter
Sample
source_name
445a
sarcoma type
WDLPS
antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were treated with 0.25% trypsin, washed with PBS, and cross-linked using 1% paraformaldehyde for 10 min at 37oC. Reactions were quenched by 0.125M glycine for 5 min. Cells were then washed with PBS and stored at -80oC. Cells were thawed on ice the next day and lysed with RIPA buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 140mM NaCl, 1% Triton x-100, 0.2%SDS, 0.1% DOC) for 10 min on ice. Sonication was performed using the Branson Sonifier 250 to achieve DNA shear length of 200-500bp. Extracts were then incubated overnight with respective antibody-Dynabead (Life Technologies) mixture (previously incubated together for 1 hr at 4oC [Rabbit IgG (Abcam), H3K9me3 (Abcam)]). Immune complexes were then washed three times with RIPA buffer, once with RIPA-500 (RIPA with 500mM NaCl) and once with LiCl wash buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% DOC). Elution and de-crosslinking were performed in direct elution buffer (10mM Tris-Cl pH 8.0, 5mM EDTA, 300mM NaCl, 0.5% SDS) by incubating immune complexes at 65oC for 4-16 hr. Proteinase K (20mg/ml) and RNaseA treatment was performed and DNA cleaned up using AMPure beads (Beckman-Coulter). Sequencing library preparation was performed using NEB kit (6040) per manufacturer's instructions.