double knock-in (DKI) mouse ES cell line carrying Foxa2-Venus and Sox17-Cherry gene fusions
genotype/variation
Foxa2-Venus heterozygous;Sox17-Cherry homozygous
facs sorting markers
Foxa2-Venus-pos/Sox17-Cherry-pos
time point
d5
chip antibody
anti-H3K4me3 (Diagenode; Pab-003-050)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
1-2 million FACS-sorted cross-linked cells (1% formaldehyde, 10min RT) were lysed in 100 ul Buffer-B (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1%SDS, 1x protease inhibitors -Roche) and sonicated in a microtube (Covaris; 520045) using a Covaris S220 device until most of the DNA fragments were 200-500 base pairs long (settings: temperature 4°C, duty cycle 2%, peak incident power 105 Watts, cycles per burst 200). After shearing, lysates were centrifuged 10min, 4°C, 12000g and supernatant diluted with 900ul of Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors-Roche). 150 ul of sonicated chromatin was then incubated 4h at 4°C on a rotating wheel with 3 µg of antibody conjugated to 10 µl of magnetic beads (Dynabeads, Life Technology). Beads were washed four times with Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors-Roche) and once with Buffer-C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were re-suspended in 100µl elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) and incubated 20min at 65 °C. Supernatant was transferred to a new tube. Crosslink reversal of immunoprecipitated DNA was carried out overnight at 65°C. Then 100µl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) was added, RNA was degraded by 4μl RNase A (10mg/ml) for 1 hour at 37°C and proteins were digested with 4μl Proteinase K (10 mg/ml) at 55°C for 2 hours. Finally, DNA was isolated by phenol:chloroform:Isoamyl alcohol purification followed by ethanol precipitation. Purified DNA was used as input for library preparation. MicroPlex Library Preparation Kit v2 (Diagenode, cat. C05010012).