Determination of Shavenbaby binding behaviour on S2 cells [chipseq_svb]

Shavenbaby (Svb) transcription factor is involved in the differenciation of epidermal cells, the homeostasis of intestinal stem cell. Svb is first synthetized as a long repressor protein called SvbRepressor (SvbREP). In presence of Pri peptides Svb Repressor is clived by the proteasoome into a shorter activator protein called Svb Activator (SvbACT). We want in this study determine the binding behaviour of the two Svb forms on S2 cells. We performed ChIPseq Svb experiment in S2 cell lines which present Svb REP or SvbACT or S2 cell without Svb. The experiment was performed in cells that either did not show Svb (Ctrl) or showed either the SvbACT or SvbREP form. 2 independent replicates were done for each condition. Cells are cross-linked with 0.8% of formaldehyde for 10 min at room temperature. Cross-linking was stopped by adding glycine (2M) and cells were washed twice in cold with 1X PBS 1 mM NaBu . Cells were resuspended in 500 μL of ChIP permeation buffer (1X PBS, 0.2% Triton, 10mM NaBu), and incubated for 20 min at room temperature. Lysis Buffer no SDS (140 mM NaCl, 15 mM HEPES pH 7.6, 1 mM EDTA, 1% Triton, 0.1% NaDOC, 0.5 mM DTT, 10mM NaBU, 25X protease inhibitor) during 5min with gentle rotation at 4°C , then cells are lysed on Lysis Buffer 1% SDS (Lysis Buffer no SDS + 1% SDS, 0.5% N-lauroylsarcosine) during 30min at 4°C with gentle rotation. Chromatin was sonicated in Bioruptor (Diagenode) using high-power settings, intervals of 30s burst/pause for six cycles to obtain fragments of ≈ 250 pb. The sonicated chromatin was diluted 10 times with Lysis Buffer (no SDS) on protein low bind tubes (Eppendorf). The chromatin fraction was cleared by centrifugation (16000 g, 10 °C, 5 min), then pre-clarified (incubed with empty beads) O/N at 4° C. Beads were washed and blocked with 0.1 mg/mL BSA in Lysis Buffer 0.1% SDS, 0.5% N- lauroylsarcosyl during 2hours at 4°C with gentle rotation. Beads are then bound to the antibody O/N at 4°C with gentle rotation. For one immunoprecipitation 20μL of blocked beads, 5μL of antibody and 400μL of Lysis Buffer 0.1% SDS (Lysis Buffer no SDS + 0.1% SDS, 0.5% N- lauroylsarcosine). Immunoprecipitation was performed for 4 hours at 4°C with gentle rotation with 5μl antibody, by adding 100μL pre-washed beads to 500μL cleared chromatin in protein low bind tubes. 2% of each sample was kept as input. Chromatin-bead complexes were eluted twice in the same tube by incubating the beads for 20 minutes at 70 °C first in 10 mM EDTA, 1% SDS, 50 mM Tris-Cl ph8, and then with TE, 0.67% SDS. Samples were incubated overnight at 65°C to reverse the cross-link. Decrosslinked samples were treated with 1μL RNAse A during 45min at 37°C then with proteinase K solution (35mg/sample glycogen,100μg/sample proteinase K) during 2h at 55°C. DNA fragments were purified by phenol- chloroform protocol and resuspended in TE.) ChIPseq of Control, SvbREP and SvbACT S2 cells (single-end 50 nt) were performed with HiSeq 200 (Illumina) at BGI.