Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Toshitsugu Fujita; Hodaka Fujii

doi:10.3791/53478

JoVE Journal > Biology

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Biyoloji

具体的基因组区域及相关分子鉴定enChIP隔离

Published: January 20, 2016

doi:

10.3791/53478

Toshitsugu Fujita¹, Hodaka Fujii¹

¹Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases,Osaka University

Özet

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Abstract

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

Introduction

与感兴趣的特定基因组区域相关联的分子的识别，需要理解的基因组功能，例如转录和表观遗传调控调节的机制。虽然几种技术已经开发了用于特定的基因组区域^1-7中的生化分析，它们没有被广泛使用，因为其固有的问题，如有限的应用程序（例如，只对于高拷贝数或基因座基因座与重复），并在此阶段太多的时间和努力需要。

为了容易地执行特定的基因组区域的生化分析，我们已经开发了两种基因座特异性染色质免疫沉淀技术，即插入的ChIP（Internet控制器^）8-13和工程化DNA结合分子介导的^{ChIP（enChIP）14-17} 。在Internet控制器，感兴趣的基因座是由标记的外源DNA结合蛋白如莱克斯插入识别序列答轨迹然后通过使用标记的DNA结合蛋白的亲和纯化分离。在enChIP，工程化DNA结合分子，如锌指蛋白，转录激活物状（TAL）的蛋白质，和群集定期相互间隔短回文重复序列（CRISPR）配合物，用于标记的感兴趣位点（ 图1）。随后，将基因组区域被标记的DNA结合分子的亲和纯化分离。

之一的enChIP过Internet控制器的一个优点是，插入的外源DNA结合蛋白的识别序列是没有必要的。靶向使用CRISPR络合物选自Cas9（dCas9）的无催化活性的形式的和导向的RNA（gRNA）位点比这些区域通过Internet控制器或enChIP使用TAL和锌指蛋白靶向容易得多。这里，我们描述了一步一步的协议enChIP与质谱联用和RNA测序（RNA片段）识别位点Associa大泰德蛋白质和RNA的分别。

Protocol

工程DNA结合分子认识到目标轨迹的1.设计对于使用CRISPR复合enChIP，使用CRISPRdirect网络工具（http://crispr.dbcls.jp），以确定所关注形容为先前18的基因组区域的候选gRNA靶序列。此Web工具将返回表格23 bp的基因组位点5'-N 20 NGG-3'的目标区域内。合成一个gBlock包括U6启动子序列和5'- N 20中5'-N 20 NGG-3'使用商业服务的序列（见图 2）19,20。用于亚克隆的目的，包括gBlock以外适当的限制性酶位点。插入gBlock到适当的载体如先前所述16。对于gBlocks几个逆转录病毒载体（请参阅材料）。对于使用TAL蛋白enChIP，设计TAL蛋白识别TARG等位点。生成质粒编码使用商业服务TAL蛋白。产生含有稠合有一个或多个标记，如3×FLAG如前所述15的TAL蛋白质的表达载体。 2.建立细胞的enChIP分析表达3×FLAG-dCas9和gRNA（CRISPR基于程序）或3×FLAG-TAL（TAL蛋白质基过程）在细胞中被如先前所描述的14-17进行分析。使用瞬时转染，如果转染效率是高的。可含有3×FLAG-dCas9和CMV启动子的表达载体（见材料）。考虑建立稳定的转化使用常规方法，如果瞬时转染效率是低的。除了为gBlock上述逆转录病毒载体，是3×FLAG-dCas9的反转录病毒载体可用（参见材料）。 <li>通过使用如先前14-17描述的抗FLAG抗体（抗体）免疫印迹分析确认3XFLAG-dCas9或3XFLAG-TAL蛋白在转染细胞中的表达。注意：这些标记的蛋白质的表达，也可以通过细胞内染色用抗FLAG-FITC和随后的FACS分析证实。一种协议，用于细胞内染色可以从我们的主页（http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/iChIP_protocols/english.html）下载。确认gRNA通过标准的RT-PCR法表达。蛋白质与感兴趣的基因组区域相互作用的定量识别，可以考虑使用稳定同位素标记的通过在细胞培养物（SILAC）分析结合enChIP（enChIP-SILAC）氨基酸。注：媒体的SILAC分析，可以从商业供应商处购买。 SILAC是在检测特异性相互作用强大。培养细胞SILAC重介质。准备细胞文化前进红色在SILAC光介质作为阴性对照。使用至少5×10 7个细胞的每个SILAC介质（重或轻）。为有效的标记，同时添加L-赖氨酸2HCl的，13 C 6和L-精氨酸盐酸，在SILAC重媒体13 C 6，15 N 4。注：至少有五细胞分裂所必需的标签蛋白。例如，培养人纤维肉瘤HT1080细胞稳定表达3XFLAG-dCas9和gRNA在37℃和5％的CO 2在DMEM培养基SILAC和透析的胎牛血清与赖氨酸2HCl的加L-精氨酸盐酸（光介质）或13 C 6 L-赖氨酸二盐酸盐加13 C 6 15 N 4 L-精氨酸盐酸（重介质）（见材料）16。添加50毫克轻或重的L-赖氨酸-2HCl的和L-精氨酸盐酸在500毫升培养基中。 Trypsinize和replate细胞才汇合，使细胞保持指数growtH。 3.交联细胞与甲醛对细胞悬浮培养，转移2×10 7细胞表达3XFLAG-TAL蛋白质或3XFLAG-dCas9加gRNA和暂停在30ml常规培养基在50ml离心管中。当使用更多的细胞，提高试剂的数量和金额比例。对于SILAC实验，混合在重介质或光介质（各5×10 7个细胞）培养的细胞和1×10 8个细胞的总分为5试管含有2×10 7细胞。添加810微升37％的甲醛的30毫升细胞悬浮液（最终浓度1％），并在37℃下5分钟。对于贴壁细胞，直接通过加入810微升37％的甲醛至30ml培养基中固定的细胞在培养皿中。停止交联通过加入3.1毫升1.25M的甘氨酸溶液（最终浓度127毫摩尔），和在室温下孵育10分钟。通过离心（300×g下5分钟，4℃）收集细胞。小心弃去上清液，包括甲醛，并将其存储在适当的废液瓶。对于贴壁细胞，分离细胞用细胞刮刀和收获在50ml管中，并作为本步骤中所述收集细胞。对于SILAC实验，混合在重介质或光介质（5×10 7个细胞每）培养分离的细胞，1×10 8个细胞的总分为5试管含有2×10 7细胞，并如在本描述的收集细胞步骤。每管洗细胞沉淀用30ml的PBS两次。小心弃去上清液，包括甲醛，并根据化学品的安全指导方针将其存储在bottle.Handle甲醛废适当的废物。固定的细胞可以冷冻并储存于-80℃。 4.制备染色质（每2×107细胞）暂停固定的细胞在10毫升细胞裂解缓冲液（10mM的Tris（pH 8.0）中，1毫摩尔EDTA，0.5％IGEPAL CA-630，和1×蛋白酶抑制剂），并在冰上孵育10分钟。离心样品（830×g下在4℃，8分钟），小心弃去上清液。暂停沉淀在10ml核裂解缓冲液（10毫摩尔Tris（pH值8.0），1mM EDTA中，0.5M氯化钠，1％的Triton X-100，0.5％脱氧胆酸钠，0.5％月桂酰肌氨酸，和1x蛋白酶抑制剂）。冰上孵育10分钟，并涡旋振荡每2-3分钟。离心样品（830× 在4℃，8分钟g），并小心地弃去上清液。在10ml PBS中洗涤沉淀。将沉淀（染色质级分）可以储存在-80℃之后在液态氮立即冻结。 5.超声处理的染色质（每2×10 7细胞）暂停在800微升的染色质部分改进的裂解缓冲液3（10毫摩尔Tris（pH值8.0），1mM EDTA中，150 mM氯化钠，0.1％脱氧胆酸钠，0.1％SDS中，和1x蛋白酶抑制剂），并转移到1.5ml试管。超声处理使用超声波仪（参见材料）的染色质和条件如下：输出：3;税：100％（连续）;时间：自由。执行超声处理10-18个循环持续10秒和在冰上冷却20秒。为了避免过热，孵育样品在冰上2分钟，每5-6个周期。为了避免起泡，保持声处理探针0.5厘米管子的底部上方的尖端的位置。离心样品（16,000×g下在4℃下进行10分钟），将上清转移到1.5ml试管。将超声处理的染色质可以储存在-80℃之后在液态氮立即冻结。 6.评估染色质碎片的混合10微升片段化的染色质与85微升蒸馏水。加入4微升和的5 M氯化钠孵育在65℃CO / N。加入1微升10毫克/毫升RNA酶A和孵育在37℃下45分钟。加入2微升的0.5M EDTA（pH 8.0）中，4微升的1M Tris（pH6.8）中，和1微升20mg / ml的蛋白酶K，再孵育在45℃下1.5小时。单独的10微升样品通过电泳中不包含染色染料，如SYBR绿1％琼脂糖凝胶。染色凝胶来评价零散染色质的长度的分布。产生0.5-2 kbp的（范围0.2-4千碱基对）的平均长度条件，推荐。纯化通过苯酚-氯仿提取或通过使用DNA提取试剂盒（参见材料）从其余样品的DNA。将纯化的DNA可以作为输入的DNA来估计enChIP分析的产率（见8.11）。 7.制备磁珠共轭与抗体（每2×10 7细胞）预削2 1.5 ml的管，一个用于使用正常小鼠IgG预澄清，另一个用于孵育抗FLAG抗体。添加150微升蛋白G偶联的磁珠（参见材料），以每管。将管上的磁体支架，等待3分钟。弃去上清液吹打。暂停在1ml PBS中含有0.01％Tween-20（PBS-T）的珠。将管上的磁性底座，等待2分钟。弃去上清液吹打，然后重复步骤。暂停在1ml含有0.1％BSA的PBS-T中的珠子。加入15微克正常小鼠IgG或抗FLAG抗体和旋转4°CO / N。后短暂离心，然后将管上的磁体支架，等待3分钟。弃去上清液吹打。暂停在1ml的PBS-T中的珠子。反转样品几次后短暂离心。将管上的磁体支架，等待3分钟，然后通过移液丢弃上清液。重复洗两次用PBS-T（总共三个洗涤步骤）。 8.染色质免疫沉淀（每2×10 7细胞）混合在5.3中制备的分散的染色质）（约800微升）与五分之一体积的（大约200微升含5％的Triton X-100（1％的Triton X-100的最终浓度）改进的裂解缓冲液3）。添加染色质溶液，以在其中结合有正常小鼠IgG蛋白G偶联的磁珠制备的管。旋转，在4℃下进行1小时。将管上的磁体支架，等待3分钟。将上清液转移到在其中结合有抗FLAG抗体蛋白G偶联的磁珠制备的管。旋转，在4°CO / N。将管上的磁体支架，等待3分钟。弃去上清液吹打。暂停在1ml的低盐缓冲液（20mM的Tris（pH 8.0）中，2毫摩尔EDTA，150毫摩尔NaCl，1％三珠吨X-100，0.1％SDS中，和1x蛋白酶抑制剂），并旋转，在4℃下5分钟。将管上的磁体支架，等待3分钟。弃上清，吹打，重复清洗步骤。洗用高盐缓冲液（20mM的Tris（pH 8.0）中，2毫摩尔EDTA，500毫摩尔NaCl，1％的Triton X-100，0.1％SDS中，和1x蛋白酶抑制剂）的两倍珠。洗用的LiCl缓冲液（10mM的Tris（pH 8.0）中，1毫摩尔EDTA，250毫的LiCl，0.5％IGEPAL CA-630，0.5％脱氧胆酸钠，和1x蛋白酶抑制剂）的两倍珠。洗用TBS-IGEPAL CA-630（50毫摩尔Tris（pH值7.5），150 mM氯化钠，0.1％IGEPAL CA-630，和1x蛋白酶抑制剂）一旦珠。暂停在200μl洗脱缓冲液的珠（50毫摩尔Tris（pH值7.5），150 mM氯化钠，0.1％IGEPAL CA-630，1×蛋白酶抑制剂，和500微克/毫升3×FLAG肽）和在37℃下20分钟。将管上的磁体支架，等待3分钟。将上清液转移到一个新的1.5毫升管，并重复ELUT离子一步。净化洗脱液通过苯酚-氯仿提取或通过使用DNA提取试剂盒的DNA小部分（例如，5％）（见材料）。将纯化的DNA可用于PCR用特定引物集来估计产量enChIP分析由与如先前14,16所述制备步骤6.7输入的DNA进行比较。 9. SDS-PAGE，染色，质谱分析混合洗脱物（400微升）用1毫升2-丙醇，加入50μl3M乙酸钠（pH 5.2）和5微升20毫克/毫升糖原。沉淀的染色质在-20℃CO / N。离心样品（16,000×g下在4℃下30分钟）并弃去上清液。冲洗用1ml 70％乙醇，离心再次的粒料（16,000×g下在4℃下进行10分钟）。吹打完全弃上清。暂停在40微升2×样品缓冲液（125毫摩尔Tris（pH6.8）中，10％的2-MERC沉淀aptoethanol，4％SDS，10％蔗糖和0.004％溴酚蓝）。涡旋5分钟，以完全溶解沉淀，然后孵育在100℃下30分钟以变性蛋白和逆转交联。对于SDS-PAGE凝胶上的运行40微升样品直至染料达到1厘米以下的井。注意：我们通常使用4-20％梯度凝胶，但其他％凝胶可以使用。染色用考马斯亮蓝或银染的凝胶。切胶成五片（2 mm高）。如先前13-16说明进行凝胶消化和质谱分析。对于SILAC实验用5×10 7个细胞每（总共1×10 8个细胞），从最初的5管在40微升2×样品缓冲液（这是没有必要按比例增加）暂停粒料。 10.纯化RNA和RNA测序分析为了净化enChIP后的RNA，加酶抑制剂的5单位/毫升（SEë材料），以所有的缓冲溶液，除改进的裂解缓冲液3和洗脱缓冲液中，向其中添加RNA酶抑制剂的40单位/毫升。混合洗脱物（400微升），16微升的5M NaCl和孵育在65℃下2小时。加入1毫升酸 – 胍 – 酚系试剂（参见材料）到样品中。涡旋15秒，然后在室温下孵育5-15分钟。离心样品15分钟，12000×g下和室温。上清转移到一个新的1.5毫升管，加入5微升对溴苯甲醚的。涡旋15秒，然后在室温下孵育3-5分钟。离心样品以12,000×克和RT 10分钟。转移上清液（约1毫升）到新的2ml管中，加入1毫升2-丙醇。倒置该管并在室温下孵育10分钟。装载到混合物中的RNA纯化试剂盒的柱（参见材料）。离心列在12000×1分钟 G和RT。洗用400μl的RNA洗涤缓冲液中的RNA纯化试剂盒（参见材料）的列。的5微升DNA酶I（1U /微升），8微升10×DNA酶I反应缓冲液，3微升DNA酶/无RNase水，和64微升的RNA洗涤缓冲液中添加80μl的DNA酶I的鸡尾酒（混合物的RNA纯化试剂盒（参见材料））到柱上。孵育样品在37℃下进行15分钟，然后在12000×g下和RT离心30秒。用400微升RNA预洗缓冲液中的RNA纯化试剂盒（见材料）的两倍洗列。洗脱用50μlDNA酶/无RNase水的核糖核酸。将洗脱的RNA可用于RNA测序。

Representative Results

总体而言，靶基因组区域的1-30％可使用enChIP纯化。图3包括代表数据表示enChIP的产率分析靶向端粒和干扰素（IFN）调节因子1（IRF-1）基因的启动子。作为典型的结果，表1列出实施例与在IRF-1启动子中鉴定由enChIP-SILAC，表一的IFNγ特异性方式相关的蛋白2列出确定enChIP与质谱联用端粒结合蛋白（enChIP-MS），和表3列出用确定enChIP-RNA测序端粒相关联的的RNA。图 enChIP 1.概述。 enChIP使用CRISPR（A，B）和 TAL（C，D，E </STRONG>）显示。感兴趣的轨迹被打上了设计DNA结合分子，如蛋白质TAL或由Cas9（DCAS）的催化活性形式的CRISPR系统和引导RNA（gRNA）。的分子间的相互作用是用甲醛固定或其它交联剂，如果需要的话。随后，固定的染色质通过超声处理或酶促消化分段。加标签的基因组区域是由亲和纯化来纯化。最后，交联逆转，相互作用的分子（基因组区域，RNA的，和蛋白质）均采用新一代测序和质谱鉴定。请点击此处查看该图的放大版本。图2.序列gBlock的。U6启动子（黑色），对G核苷酸b安伏的导向序列（蓝色），引导序列（gRNA间隔物）（红色），所述支架序列（绿色），及终止子序列（橙色）被示出。图3.产量的代表enChIP分析。（A）中的百分比的输入为目标的IRF-1基因座和非靶Sox2的基因座。（B）的百分比的输入为目标端粒和非目标γ-卫星。该数字已调整，并从以前的出版物15,16修改。分类蛋白质转录 DDX1，PARP1，CKAP4，Pescadillo同源，PURβ，活化的RNA聚合酶II转录激活p15基因， BTF3，Myb的结合蛋白1A 组蛋白脱乙酰化，辅阻遏物成分 RBBP4，PA2G4，TBL3 乙酰转移酶蛋白质精氨酸N-甲基转移酶1 DNA拓扑异构酶 DNA拓扑异构酶2α 组蛋白组蛋白H2A.Z，组蛋白H3.2 表1：用在确定enChIP-SILAC一个IFNγ特异性方式的人IRF-1的启动子区相关的蛋白质的例子的表已被改编从先前的出版物16。分类蛋白质哺乳动物端粒结合蛋白 PML，RPA，CDK1，PARP1，PCBP1 端粒碧在酵母nding蛋白质或其他生物 IMP4 与蛋白质相互作用的端粒结合蛋白质[相关端粒结合蛋白] DNA聚合酶α（POLA1）Cdc13p] ARMC6 [TRF2]，CTBP1 [FOXP2-POT1] exportin-5 [叔]，GNL3L [TRF1] exportin-1 [叔]，14-3-3 [叔] 蛋白定位于异 BEND3 蛋白质调控的表观遗传标记 KDM5C 蛋白质的突变影响端粒功能 DNA聚合酶α（POLA1），HAT1，Nup133， CDK7，DPOE1，PRDX1，TYSY，谷氨酸半胱氨酸连接酶，谷，SMRC1 表2：用确定enChIP-MS鼠标端粒相关蛋白的例子表已adapted从以前的出版物15。分类 RNA的端粒酶组件 TERC，RMRP 端粒的RNA TERRAS scaRNAs Scarna6，Scarna10，Scarna13，Scarna2 H / ACA 52]形成 Snora23，Snora74a，Snora73b，Snora73a C / D 52]形成 Snord17，Snord15a，Snord118 lncRNA Neat1 表3：具有确定enChIP-RNA的序列表已被改编自先前公布17 鼠标端粒相关的RNA的例子。

Discussion

Here, we describe the purification of specific genomic regions using engineered DNA-binding molecules such as the CRISPR system and TAL proteins, and the identification of proteins and RNAs bound to these genomic regions. Binding of engineered DNA-binding molecules to the genome may affect chromatin structure, including nucleosome positioning, and may abrogate genomic functions, as described in CRISPR interference experiments²¹. To avoid these potential aberrant effects, we propose specific guidelines for choosing target genomic regions. First, to avoid potential inhibition of the recruitment of RNA polymerases and transcription factors, as well as disruption of nucleosome positioning around the transcription start site, the target regions for analyses of promoter regions should be several hundred base pairs upstream of (5′ to) the transcription start site. By contrast, when analyzing genomic regions with distinct boundaries, such as enhancers and silencers, genomic regions that are directly juxtaposed to these regions can be targeted because it is less likely that the binding of engineered DNA-binding molecules will affect their functions. Furthermore, it is best to avoid using target regions that are conserved among different species, because important DNA-binding molecules often bind to evolutionarily conserved regions and inhibition of their binding might disrupt the functions of the target genomic regions. In this regard, it is always necessary to check that the function of the target genomic region is maintained in the established cells used for enChIP analyses. Because multiple gRNAs can be tested easily and it is tedious and expensive to generate multiple versions of TAL or zinc-finger proteins recognizing different target genomic regions, enChIP using CRISPR is more advantageous than enChIP using other proteins.

It has been shown that dCas9 binds to off-target sites although affinity to those sites might be weaker than that to the target sites^22-25. There are several ways to manage contamination of molecules bound to those off-target sites. First, the use of several, at least two, different gRNAs would be recommended. Those molecules commonly observed in enChIP using distinct gRNAs would be true positives. Second, comparison of different conditions for enChIP would be effective in cancelling contamination of non-specific molecules and molecules bound to off-target sites. Examples of those comparison sets would be (i) stimulation (-) and (+), or (ii) different cell types such as T cells vs. B cells. Finally, quantitative analysis of binding of candidate molecules should be performed to confirm their specific binding to the target sites. It is preferable to prepare cells expressing only dCas9 but not gRNA as a negative control.

Using enChIP analyses, we were able to successfully identify a number of known and novel molecules interacting with specific genomic regions (Tables 1-3)^14-17. However, this technique failed to detect some other known proteins interacting with these regions. For example, STAT1 reportedly associates with the IRF-1 promoter upon IFNγ stimulation⁸, but our enChIP-SILAC analysis did not detect STAT1 as a protein induced to interact with this genomic region¹⁶. In addition, in the enChIP-MS analysis of telomeres, we did not detect shelterin proteins consisting of TRF-1 and TRF-2¹⁵, which have been shown to interact with telomeres²⁶. There are a few potential reasons for these discrepancies. First, the stoichiometry of binding of Stat1 to the IRF-1 promoter might be very low. It is reasonable that enChIP-MS, including enChIP-SILAC, detects proteins that are more abundantly associated with target genomic regions; hence, the analysis of more cells might be necessary to detect these proteins. Increases in the sensitivities of MS instruments would also contribute to the efficient detection of proteins with low stoichiometric binding. Second, some proteins, possibly including Stat1 and shelterins, might be difficult targets for MS analyses. Third, in our analysis of telomere-binding proteins¹⁵, the 3×FLAG-TAL proteins recognizing telomeres (3×FN-Tel-TAL) might have blocked the binding of shelterins to telomeres in a competitive fashion.

In contrast to the relative difficulty of detecting transcription factors binding to specific genomic regions using enChIP, we successfully identified epigenetic regulators such as histone modification enzymes using enChIP analyses. The success of this technique may be due to the fact that epigenetic regulators bind to a broad range of genomic regions; hence, more proteins per genomic region are available for MS. Because epigenetic regulators are increasingly recognized as important targets for drugs against intractable diseases such as cancer, enChIP would be a useful tool for the identification of epigenetic drug targets.

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

这项工作是由武田科学基金会（TF）的支持;旭硝子基金会;上原纪念基金会（HF）;在仓田纪念日立科学技术基金会（TF和HF）;格兰特提供的援助青年科学家（B）（＃25830131），格兰特 – 在急救科学研究（C）（＃15K06895）（TF）;和格兰特 – 在急救科学研究的创新领域“转录周期”（＃25118512＆＃15H01354），格兰特 – 在急救科学研究（B）（＃15H04329），格兰特提供的援助的探索性研究（＃26650059）和“基因组支持”（＃221S0002）（HF）由教育部，文化，体育，科学和技术的日本。

Materials

gBlock synthesis service	Life Technologies	Gene Synthesis by GeneArt
gBlock synthesis service	IDT (Integrated DNA Technologies)	gBlocks Gene Fragments
pSIR-neo	Addgene	51128
pSIR-GFP	Addgene	51134
pSIR-DsRed-Express2	Addgene	51135
pSIR-hCD2	Addgene	51143
TAL synthesis service	Life Technologies	GeneArt Precision TALs
3xFLAG-dCas9/pCMV-7.1	Addgene	47948
3×FLAG-dCas9/pMXs-puro	Addgene	51240
3×FLAG-dCas9/pMXs-IG	Addgene	51258
3×FLAG-dCas9/pMXs-I2	Addgene	51259
3×FLAG-dCas9/pMXs-neo	Addgene	51260
anti-FLAG M2 Ab	Sigma-Aldrich	F1804
FITC-conjugated anti-FLAG M2	Sigma-Aldrich	F4049
DMEM medium for SILAC	Life Technologies	89985	Other medium can be purchased from Life Technologies
Dialyzed FBS for SILAC	Life Technologies	89986
L-Lysine-2HCl for SILAC	Life Technologies	89987	for Light medium
L-Arginine-HCl for SILAC	Life Technologies	89989	for Light medium
L-Lysine-2HCl, 13C6 for SILAC	Life Technologies	89988	For Heavy medium
L-Arginine-HCl, 13C6, 15N4 for SILAC	Life Technologies	89990	For Heavy medium
Complete, mini, EDTA-free	Roche Diagnostics	4693159
Ultrasonic Disruptor UD-201	Tomy Seiko
ChIP DNA Clean & Concentrator	Zymo Research	D5205
Dynabeads-Protein G	Life Technologies	DB10004
RNasin Plus RNase Inhibitor	Promega	N2611
Isogen II	Nippon Gene	311-07361
Direct-zol RNA Miniprep kit	Zymo Research	R2050
LTQ Orbitrap Velos	Thermo Fisher Scientific		a component of a nanoLC-MS/MS system for MS analysis
nanoLC	Advance, Michrom Bioresources		a component of a nanoLC-MS/MS system for MS analysis
HTC-PAL autosampler	CTC Analytics		a component of a nanoLC-MS/MS system for MS analysis

Referanslar

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Etiketler

EnChIP Genomic Region Isolation Molecular Interactions Transcription Epigenetic Regulation DNA-binding Molecules FLAG-dCas9 FLAG-TAL Cross-linking Formaldehyde Glycine Chromatin Extraction Lysis Buffer Nuclear Lysis Buffer

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Fujita, T., Fujii, H. Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP. J. Vis. Exp. (107), e53478, doi:10.3791/53478 (2016).