Illumina HiSeq 4000 sequencing; ChIP-seq experiment of the beta-catenin co-factor BCL9 and the T-box transcription factor TBX3 in developing mouse forelimbs.
ChIP-seq experiment of the beta-catenin co-factor BCL9 and the T-box transcription factor TBX3 in developing mouse forelimbs.
Forelimbs buds were manually dissected from ca. 250 RjOrl:SWISS outbred 10.5 dpc mouse embryos. Chromatin immunoprecipitation was performed as in Cantù et al., 2018. Briefly, the tissue was dissociated to a single cell suspension with collagenase (1 ug/ul in PBS) for 1 hour at 37° C, washed and cross-linked in 20 ml PBS for 40 min with the addition of 1.5 mM ethylene glycol-bis(succinimidyl succinate) (Thermo Scientific, Wal- tham, MA, USA), for protein-protein cross-linking, and 1% formaldehyde for the last 20 min of incubation, to preserve DNA-protein interactions. The reaction was blocked with glycine and the cells were subsequently lysed in 1 ml hepes buffer (0.3% SDS, 1% Triton-X 100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES). Chromatin was sheared using Covaris S2 (Covaris, Woburn, MA, USA) for 8 min with the following set up: duty cycle: max, intensity: max, cycles/burst: max, mode: Power Tracking. The sonicated chromatin was diluted to 0.15% SDS and incubated overnight at 4°C with 1 ug of anti-BCL9 (Abcam, ab37305) and anti-TBX3 (Santacruz, sc17871) antibodies, or IgG and 50 ul of protein A/G magnetic beads (Upstate) per sample. The beads were washed at 4°C with wash buffer 1 (0.1% SDS, 0.1% deoxycholate, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES), wash buffer 2 (0.1% SDS, 0.1% sodium deoxycho- late, 1% Triton X-100, 0.5 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES), wash buffer 3 (0.25 M LiCl, 0.5% sodium deoxycholate, 0.5% NP-40, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES), and finally twice with Tris EDTA buffer. The chromatin was eluted with 1% SDS, 0.1 M NaHCO3, de-crosslinked by incubation at 65°C for 5 h with 200 mM NaCl, extracted with phenol-chloroform, and ethanol precipitated. The immunoprecipitated DNA was used as input material for DNA deep sequencing. Libraries were prepared starting with 5 ng of ChIP-enriched DNA as starting material and processed with the Illumina TruSeq ChIP kit according to manufacturer specifications. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies).
Experimental Factor: immunoprecipitate
Input_replicate 2