GSM1129743: PGC Input ChIPSeq; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Embryo
Cell type
Primordial germ cells
NA
NA
Attributes by original data submitter
Sample
source_name
E11.5 Primordial Germ Cells, input
strain/background
transgenic Oct4:GFP B6 crossed to Swiss-Webster
cell type
primordial germ cells (PGCs)
age
E11.5
chip antibody
none
library amplification cycles
15
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Two aliquots of E11.5 PGCs, consisting of 104,000 and 97,000 cells, were cross-linked in 0.25% formaldehyde (Thermo Scientific 28906) in PBS for 10min at room temperature prior to quenching with 125mM glycine. Cross-linked material was sonicated using a Covaris sonicator for 12' at Duty 5%, Intensity 3, and Bursts 200. Lysate for each replicate was diluted in lysis buffer and cleared of debris. Cleared lysate from each replicate was divided equally to perform ChIP for H3K4me3, H3K27me3, and an input control. For each IP, 11µl of protein A Dynabeads (Life Technologies 1001D) were pre-incubated with 2.4µg of either anti H3K4me3 (Diagenode pAb003-050, lot # A5051-001P) or H3K27me3 (Millipore 07-449, lot # 1999681) in ChIP lysis buffer. Lysates were incubated with preformed bead/antibody overnight at 4C with mixing. The chromatin/bead/antibody complexes were washed sequentially with lysis buffer three times, DOC buffer once, and TE buffer once, followed by a transfer to a fresh PCR tube. Chromatin was eluted using elution buffer (1% SDS, 0.1M NaHCO3). IP and Input samples were treated with RNaseA followed by Proteinase K treatment. Cross-linking was reversed by incubating overnight at 65C while shaking. IP-DNA and Inputs were purified using a MinElute PCR Purification Kit (Qiagen 28004). Libraries were generated using the ThruPLEX-FD Prep Kit (Rubicon Genomics R40048) with 20 cycles of amplification for IP-DNA and 15 cycles for Input DNA.