GSM2895199: Pali dKO ES H3K27me3; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K27me3
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
Embryonic Stem Cell
cell type
Embryonic Stem Cell
genotype
Pali1-/-, Pali2-/-
antibody
H3K27me3
spike-in reference cell line
Drosophila melanogaster S2
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Embryonic stem cells were washed once with PBS before crosslinking for 10 minutes with PBS containing 1% formaldehyde (Sigma). Crosslinking was quenched with 0.125M Glycine for 5 minutes before two PBS washes. The crosslinked cells were lysed in 6mL of SDS-Lysis buffer (100mM NaCl, 50mM Tris pH8.1, 5mM EDTA pH 8.0, 0.02% NaN3, 0.5% SDS, 2μg/mL Aprotonin, 1μg/mL Leupeptin, 1mM PMSF). Chromatin was pelleted by centrifugation at 1200RPM for 5 minutes at room temperature. The supernatant was then discarded and the chromatin was resuspended in 3mL of ChIP buffer (2:1 dilution of SDS-Lysis buffer: Triton dilution buffer [100mM Tris pH 8.6, 100mM NaCl, 5mM EDTA pH 8.0, 0.02% NaN3, 5% Triton X-100, 2μg/mL Aprotonin, 1μg/mL Leupeptin, 1mM PMSF]). Chromatin was sheared to approximately 100bp-1000bp fragments by successive 30 second rounds of sonication at 5-8% amplitude. Sonicated chromatin was pre-cleared for 30 minutes using equilibrated protein A beads (Sigma) that had been blocked in TE (10mM Tris pH8.1, 1mM EDTA pH 8.0) containing 0.5mg/mL BSA and 0.2mg/mL Herring Sperm DNA. 10-100µg (DNA) of chromatin was incubated overnight with antibody while rotating at 4°C. For ChIP-RX 250 µg of chromatin (protein) was used per ChIP with a 1.67% spike in of Drosophila S2 chromatin and 5µg of antibody was used per ChIP. Following clarification of insoluble precipitates the chromatin was incubated for 3 hours with 50µL of blocked protein A beads. After incubation the beads were washed three times in Mixed Micelle Buffer (150mM NaCl, 20mM Tris pH 8.1, 5mM EDTA pH 8.0, 5.2% Sucrose, 0.02% NaN3, 1% Triton X-100, 0.2% SDS), twice with Buffer 500 (0.1% Sodium Deoxycholate, 1mM EDTA pH 8.0, 50mM HEPES pH7.5, 1% Triton X-100, 0.02% NaN3), twice with LiC detergent wash (0.5% Sodium Deoxycholate, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP-40, 10mM Tris pH 8.0, 0.02% NaN3) and finally one wash with TE. Immunoprecipitated material was eluted from the beads with Elution buffer (0.1M NaHCO3, 1% SDS) while shaking for 1 hour at 65°C. The supernatant of the elution was retained and incubated overnight at 65°C while shaking to reverse the crosslinks. The eluted complexes were then subject to RNase (Thermo Fisher) and Proteinase K (Sigma) treatment prior to DNA clean up through Phenol Chloroform clean up and Ethanol precipitation. ChIP enrichment was analysed by qPCR using the SYBR Green I detection chemistry (Applied Biosystems) on the ABI Prism 7500 Fast Real–Time PCR System. For library prep, we used the NEBNext Ultra DNA library preparation kit (NEB) for library prep and the Kapa library quantification for Illumina (Roche #KK4835). Sequencing was performed with Illumina HiSeq v4 chemistry on a HiSeq 2500.