Embryonic stem cells with Drosophila spike-in, Tamoxifen treated, MNase-digested chromatin control
spiked-in organism
Drosophila melanogaster
cell type
RYBPfl/fl;YAF2-/- ES cells
treatment
tamoxifen
chip antibody
none
biological replicate
1
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
A native ChIP protocol combined with a calibrated ChIP-seq approach was employed, as previously described (Hu et al., 2015; Bonhoure et al., 2014; Orlando et al., 2014). To achieve this, 1.25 x 10^7 Drosophila melanogaster S2 cells were spiked in to 5 x 10^7 Rybpfl/fl;Yaf2-/- mouse embryonic stem cells with or without OHT treatment. Nuclei were then isolated with RSB buffer (10 mM Tris-HCl (pH 8.), 10 mM NaCl, 3 mM MgCl2) supplemented with 0.1% NP-40 and 5 mM N-ethylmaleimide, followed by MNase digestion for 5 min with 150 U MNase (Fermentas, Waltham, MA) in 1 ml RSB supplemented with 0.25 M sucrose, 3 mM CaCl2 and 10 mM N-ethylmaleimide. Digestions were stopped with EDTA, before nuclei were pelleted by centrifugation at 1500 x g and the soluble S1 fraction collected. Pelleted nuclei were then resuspended in 300 µl nucleosome release buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.2 mM EDTA, 10 mM N-ethylmaleimide), incubated at 4°C for 1 hr with gentle rotation, and then gently passed through a 27G syringe needle five times. Following centrifugation to pellet the insoluble material, the soluble S2 fraction was collected and combined with the S1 fraction. Immunoprecipitations were performed overnight at 4°C using chromatin corresponding to 5 x 10^6 cells. Antibody-bound chromatin was isolated on protein A agarose beads (RepliGen, Waltham, CA) or protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA). Native histone ChIPs were washed four times with native ChIP wash buffer (20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 125 mM NaCl and 0.1% Triton), followed by a final TE buffer wash. Samples were then treated with RNase and proteinase K before being purified with ChIP DNA Clean & Concentrator™ kit (Zymo, Irvine, CA). For calibrated ChIP-seq experiments, an input control was prepared for each individual sample, to allow the quantitation of spike-in consistency. Libraries were prepared using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® kit, with 8-10 PCR cycles. Libraries were purified using AMPure XP cleanup, and quantified by qPCR using KAPA Library Quantitation Standards (KAPA Biosystems). Libraries were sequenced as 40bp paired-end reads on Illumina NextSeq 500 platform.