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. Overall design: 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.

Shavenbaby (Svb) 转录因子（transcription factor）参与表皮细胞分化与肠道干细胞稳态维持。Svb最初以名为Svb阻遏蛋白（SvbREP）的长链阻遏蛋白形式被合成。当存在Pri肽时，Svb阻遏蛋白会被蛋白酶体（proteasome）切割为一种名为Svb激活蛋白（SvbACT）的短链激活蛋白。本研究旨在探究两种Svb亚型在S2细胞中的结合特性。我们在分别表达SvbREP、SvbACT以及不表达Svb的S2细胞系中开展了Svb的染色质免疫沉淀测序（ChIP-seq）实验。

实验设计概述：本实验的细胞分为三组：不表达Svb的对照组（Ctrl）、表达SvbACT的实验组与表达SvbREP的实验组，每组均设置2次独立生物学重复。

具体实验流程如下：细胞采用0.8%甲醛在室温下交联10分钟，随后加入2M甘氨酸终止交联，并用预冷的1×PBS（含1mM丁酸钠（NaBu））洗涤细胞两次。将细胞重悬于500 μL ChIP渗透缓冲液（1×PBS、0.2% Triton X-100、10mM丁酸钠（NaBu））中，室温孵育20分钟。使用不含SDS的裂解缓冲液（140mM NaCl、15mM HEPES pH 7.6、1mM EDTA、1% Triton X-100、0.1%脱氧胆酸钠（NaDOC）、0.5mM DTT、10mM丁酸钠（NaBu）、25×蛋白酶抑制剂混合物），4℃轻柔旋转孵育5分钟；随后更换含1% SDS的裂解缓冲液（即上述不含SDS的裂解缓冲液添加1% SDS与0.5% N-月桂酰肌氨酸），4℃轻柔旋转裂解30分钟。将染色质置于Diagenode Bioruptor超声破碎仪中，采用高功率模式进行超声破碎：以30秒超声/30秒暂停为一个循环，共6个循环，以获得约250 bp的染色质片段。将超声破碎后的染色质用不含SDS的裂解缓冲液按10倍比例稀释至低蛋白结合离心管（Eppendorf）中，随后以16000 g、10℃离心5分钟以澄清染色质组分，接着加入空白磁珠进行预澄清，4℃孵育过夜。将磁珠用含0.1mg/mL牛血清白蛋白（BSA）的0.1% SDS裂解缓冲液（含0.5% N-月桂酰肌氨酸）在4℃轻柔旋转孵育2小时以洗涤并封闭磁珠。随后将磁珠与抗体在4℃轻柔旋转孵育过夜以结合抗体。单次免疫沉淀反应体系为：20μL封闭后的磁珠、5μL抗体、400μL 0.1% SDS裂解缓冲液（即不含SDS的裂解缓冲液添加0.1% SDS与0.5% N-月桂酰肌氨酸）。取5μL抗体，加入500μL澄清后的染色质与100μL预洗涤的磁珠，在4℃轻柔旋转孵育4小时以完成免疫沉淀。每个样品留存2%作为Input对照。将染色质-磁珠复合物在同一管中进行两次洗脱：首先加入含10mM EDTA、1% SDS、50mM Tris-Cl pH 8的洗脱液，70℃孵育20分钟；随后加入TE缓冲液（含0.67% SDS）继续孵育。将洗脱样品于65℃孵育过夜以逆转交联。脱交联后的样品先用1μL核糖核酸酶A（RNase A）在37℃处理45分钟，随后加入蛋白酶K（proteinase K）溶液（每样品含35mg糖原（glycogen）、100μg蛋白酶K）在55℃孵育2小时。采用酚-氯仿法纯化DNA片段，最终用TE缓冲液重悬。

本研究中对照组、SvbREP组与SvbACT组S2细胞的ChIP-seq实验采用单端50 nt测序策略，由华大基因（BGI）在Illumina HiSeq 200测序仪上完成。