GSM4147297: Auxin G2 input; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
HeLa S3 cells with a homozygous knock-in of the AID tag to the WAPL gene
cell line
HeLa S3
genotype/variation
homozygous knock-in of the AID tag to the WAPL gene
cell cycle phase
G2
chip antibody
none (input)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
G2 and metaphase arrested HeLa S3 cells were collected and fixed in 1xPBS containing 1% formaldehyde for 10 minutes. Glycine was added to a final concentration of 0.125M to stop cross-linking and cells washed in cold 1xPBS. Intact nuclei from ~ 5x106 cells were extracted with 1ml of Farnham lysis buffer and finally resuspended into 300ml of RIPA buffer. Nuclei were fragmented using a Qsonica sonicator for 5 minutes (30 seconds on/off, 40%). The efficiency of fragmentation was measured as the enrichment of ~ 300 bp fragments, tested on an agarose gel, and the DNA quantified in a Biodrop. To perform immunoprecipitations Dynabeads M-280 Sheep Anti-Rabbit (Invitrogen 11203D) were coupled to 5 mg of pS2 or IgG rabbit antibodies and incubated with 25 mg of fragmented chromatin samples overnight at 4°. Beads were then washed with LiCl wash buffer and eluted in 200 ml of IP elution buffer. Cross-link was reversed by incubating beads at 65° for 1 hour, supernatant (DNA) recovered after centrifugation and incubated at 65° for 14h. In parallel, input samples (25 mg of fragmented chromatin) were incubated at 65° for 14h and then purified using QIAquick PCR Purification Kit (QIAGEN). For ChIP-seq, DNA recovered from pS2 IP and ~ 10 ng of sonicated INPUTs were used for library preparation NEBNext ChIP-Seq kit (NEB6240S)