Cell line integrating with FLXist randomly on Chr3 (P4D7F4)
cell type
mESC
spiked-in
Drosophila
agent
Dox
time
24h
antibody
H2AK119ub (CST #8240S, rabbit mAb D27C4)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For calibrated native ChIP (H3K27me3 and H2AK119ub), 40 million ES cells and 10 million Drosophila SG4 Cells were pooled and lysed in RSB (10 mM Tris pH 8, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40) for 5 min on ice with gentle inversion. Nuclei were resuspended in 1 ml of RSB + 0.25 M sucrose + 3mM CaCl2, treated with 200U of MNase (Fermentas) for 5 minutes at 37ºC, quenched with 4 µl of 1M EDTA, then centrifuged at 5000 rpm for 5 minutes. The supernatant was transferred to a fresh tube as fraction S1. The chromatin pellet was incubated for 1 h in 300 µl of nucleosome release buffer (10 mM Tris pH 7.5, 10 mM NaCl, 0.2 mM EDTA), carefully passed 5 times through a 27G needle then centrifuged at 5000 rpm for 5 minutes. The supernatant from this S2 fraction was combined with S1. For each ChIP reaction, 80 µl of chromatin was diluted in Native ChIP incubation buffer (10 mM Tris pH 7.5, 70 mM NaCl, 2 mM MgCl2, 2 mM EDTA, 0.1% Triton) to 1 ml and incubated with antibody overnight at 4ºC. Samples were incubated with 50 µl protein A agarose beads preblocked in Native ChIP incubation buffer with 1 mg/ml BSA and 1 mg/ml yeast tRNA, then washed for a total of 4 times in Native ChIP wash buffer (20 mM Tris pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton) and once in TE. The DNA was eluted with 1% SDS and 100 mM NaHCO3, and was purified using ChIP DNA Clean and Concentrator kit (Zymo). NEBNext Ultra II DNA Library Prep Kit