title: ChIP-Seq of Sall4 genetically modified mouse embryonic stem cells compared to wild type controls
design: ChIP-Seq of Sall4 genetically modified mouse embryonic stem cells compared to wild type controls
library_construction_protocol: The cells used are mouse embryonic stem cells. The antibody used for ChIP-Seq is rabbit- α-SALL4 (ab29112, Abcam) Cells were fixed for 15 mins by addition of 36.5% methanol-stabilised formaldehyde solution and cross-linking was quenched in 125 mM glycine before cells were washed and harvested in ice-cold PBS. Cells were lysed with cell lysis buffer (50mM Tris-HCl pH8, 2mM EDTA pH8, 0.1% NP40, 10% glycerol) and incubated for 10 mins on ice. The lysed cells were then centrifuged to pellet nuclei. Nuclear pellets were resuspended in 1% SDS, 10mM EDTA, 50mM Tris-HCl pH8 and fragmented by sonication. Chromatin (100 μg) was pre-cleared with A/G magnetic beads (ThermoFisher Scientific Scientific) for 1 hr at 4°C and then immuno-precipitated in 0.5%NP40, 200mM NaCl, 50mM Tris-HCl pH8 with 7μg of antibody overnight. Drosophila melanogaster spike-in chromatin and antibody (Active Motif) were added for normalisation, according to the manufacturer's instructions. Washes were performed using low salt (0.1% SDS, 0.5% NP40, 2mM EDTA, 20mM Tris-HCl pH8, 150mM NaCl), high salt (0.1% SDS, 0.5% NP40, 2mM EDTA, 20mM Tris pH8, 500mM NaCl) and LiCl buffer (250mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1mM EDTA, 20mM Tris-HCl pH8) and eluted samples were reverse-crosslinked for >4 hr at 65°C with 0.3 M NaCl and 3 μg/ml RNase A (Roche). Proteinase K treatment was performed for 2 hr at 45°C with 10 mM EDTA, 40 mM Tris-HCl pH 6.5 and 20 μg proteinase K. Chromatin was purified from two independent biological replicates using SPRI beads (Beckman Coulter) and DNA concentrations determined using Qubit fluorometric quantitation (ThermoFisher Scientific).  Libraries were prepared using the NEBNext Ultra II DNA library kit (NEB). Index Primers were used to multiplex libraries.