GSM2845190: BiotinChIP_FoxD3_ChIP; Danio rerio; ChIP-Seq
FACS sorted cells were washed with PBS and stored at -80C in lysis buffer. RNA was extracted using Ambion RNAqueous Micro Total RNA isolation kit (AM1931), checked on Bioanalyser, samples with RIN >7 were used to prepare cDNA using Takara Clontech SmartSeq2 V4 kit (634889). Sequencing libraries were prepared using Illumina Nextera XT library preparation kit (FC-131-1024). Foxd3 Biotin-ChIP was performed on 320 5-6ss whole embryos (128,000 cells of interest). Embryos were manually dechorionated, cells were dissociated with 20 strokes using pestle A in isotonic nuclei extraction buffer (NEB: 0.5% NP40, 0.25% Triton X, 10 mM Tris-HCl (pH 7.5), 3 mM CaCl2, 0.25 M sucrose, 1mM DTT, 0.2 mM PMSF, 1X Proteinase inhibitor (PI) ) in a glass homogeniser and cross-linked using 1% formaldehyde at room temperature for 10 min. Fixation was quenched with 125 mM of glycine for 5min. Cross-linker was washed-out by 3x pellet washes with 1x PBS (with 1X PI, 1 mM DTT and 0.2 mM PMSF) centrifuging at 2000 x g for 4min at 4C. Pellets were re-suspended in NEB. Cell nuclei were expulsed with 20 strokes using pestle B in a glass homogeniser, pelleted and washed with 1 xPBS (with 1X PI, 1 mM DTT and 0.2 mM PMSF). Nuclei were lysed in SDS lysis buffer (0.7% SDS, 10mM EDTA, 50 mM Tris-HCl (pH 7.5), 1x PI). Cross-linked chromatin was sonicated at 12A 10x (10s ON 30s OFF) followed by 8A 4x (30s ON 30s OFF). Sheared chromatin samples were pre-cleared in pre-blocked Protein G beads (Dynabeads Protein G, Life Technologies) for 1 hour at 4C. 1/20 of biotinChIP was collected as an input fraction and stored at -80C. Pre-cleared chromatin samples were incubated on pre-blocked streptavidin beads (Dynabeads M-280 streptavidin beads, Life Technologies) o/n at 4C. Beads were washed with SDS Wash Buffer (2% SDS, 10mM Tris-HCl (pH 7.5), 1 mM EDTA) at room temperature, followed by 4x RIPA washes (50 mM Hepes-KOH (pH 8.0), 500 mM LiCl, 1mM EDTA, 1% NP40, 0.7% Na-Deoxycholate, 1x PI) and 1x Na-Cl TE wash (1x TE, 50mM NaCl) at 4C. Chromatin was eluted from the beads with SDS ChIP elution buffer (50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% SDS). Cross-linking was reversed o/n at 70C in the thermomixer at 1300 rpm. Cellular RNA was digested with RNAse A (0.2 g/ml) at 37C for 1 hour, and cellular protein with Proteinase K (0.4 mg/ml) at 55C for 2 hours. Chromatin samples were separated from the streptavidin beads. Input and ChIP DNA was extracted using a standard phenol-chloroform extraction method. Libraries were prepared using MicropPlex Library Preparation kit (Diagenode) and sequenced using NextSeq 500/550 High Output Kit v2 (75 cycles) on NextSeq 500 sequencing system.