Mapping of ETV1 genomic binding sites in gastrointestinal stromal tumor (GIST).. Homo sapiens

ETV1 is highly expressed in GIST and their presumed precursors--the interstitial cells of Cajal. ETV1 is required for survival for both GIST and ICC and regulates GIST signature genes. Here, using Illumina-Solexa based next-generation sequencing of ETV1 chromatin immunoprecipates (ChIP-Seq), we define ETV1 binding sites in the GIST48 cell line. Overall design: Crosslinked ChIP using input control ETV1 ChIP in GIST48 cells. Details: GIST48 cells were crosslinked for 15-minutes in 1% parformaldehyde. Cells were lysed and chromatin sheared using bioruptor. Sheared chromatin was incubated with anti-rabbit IgG dynabeads pre-conjugated with anti-ETV1 antibody (Abcam Ab81086, Lot 787879), washed, eluted, reverse cross-linked, and purified. Purified ChIP DNA was blunt-ended, ligated to Solexa adaptors, amplified with 18-cycles of PCR, and sequenced on Solexa Genome Analyzer. All reads (~36-bp) from ChIP-Seq were aligned to the human genome (build 36, or hg18) using the ELAND alignment software within the Illumina Analysis Pipeline. Unique reads mapped to a single best-matching location with no more than two mismatches were kept and used to generate genome-wide distribution of ETV1-binding and for peak identification. Redundant reads were considered once.

ETV1在胃肠道间质瘤（Gastrointestinal Stromal Tumor, GIST）及其假定前体——卡哈尔间质细胞（Interstitial Cells of Cajal, ICC）中呈高表达。ETV1对于GIST和ICC的存活均不可或缺，并调控GIST的特征性基因。本研究通过基于Illumina-Solexa的下一代测序技术对ETV1染色质免疫沉淀产物进行测序（ChIP-Seq），明确了GIST48细胞系中ETV1的结合位点。

实验总体设计：采用交联染色质免疫沉淀技术，以输入样本作为对照，在GIST48细胞中开展ETV1染色质免疫沉淀实验。

具体实验流程：将GIST48细胞在1%多聚甲醛中交联15分钟；裂解细胞后，使用Bioruptor超声破碎仪进行染色质剪切；将剪切后的染色质与结合有抗ETV1抗体（Abcam公司货号Ab81086，批次787879）的抗兔IgG磁珠孵育，随后经洗涤、洗脱、逆转交联并纯化；将纯化得到的ChIP DNA进行末端补平，连接Solexa接头，通过18个循环的PCR进行扩增，最终在Solexa基因组分析仪上完成测序。

数据分析：所有来自ChIP-Seq的长度约36bp的测序reads，均通过Illumina分析流程内置的ELAND比对软件比对至人类基因组（版本36，即hg18）。仅保留比对至单个最佳匹配位点且错配数不超过2个的唯一比对reads，用于绘制ETV1结合位点的全基因组分布图谱并进行结合峰识别；冗余reads仅统计一次。