RING1A-/-;RING1Bfl/fl Embryonic stem cells, untreated control, sonicated control
cell type
Ring1A(-/-);Ring1B(fl/fl);R26::CreERT2 ES cells
passage
15-25
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
To prepare material for ChIP-seq, immunoprecipitation was performed overnight at 4°C with approximately 3 µg of antibody and chromatin corresponding to 5 × 10^6 cells. Antibody bound chromatin was isolated on protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA) and washes were performed with low salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP40, 1% Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)) and TE buffer (x2) (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). To prepare ChIP-seq material, ChIP DNA was eluted, and cross-links reversed at 65°C, then samples were then sequentially treated with RNase and proteinase K before being purified on a PureLink PCR micro column (Invitrogen). ChIP sequencing libraries were generated as described previously (Blackledge et al., 2010) and sequenced on the Illumina HiSeq2000 platform with 51 bp reads. ChIP sequencing experiments were performed in biological duplicates with matched input controls. Performed by the High-Throughput Sequencing centre at the Wellcome Trust Centre for Human Genetics (Oxford, United Kingdom) according to standard Illumina library generation protocol.