ena-DATASET-ErasmusMC-hema-25-04-2021-16:58:30:197-1306 - samples

ChIP-seq data were generated for a number of selected patients to investigate changes in enhancer and promoter regions. ChIP was performed as described previously with slight modifications27. Briefly, cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature and the reaction was quenched with glycine at a final concentration of 0.125 M. Chromatin was sheared using the Covaris S220 focused-ultrasonicator to an average size of 250–350 bp. A total of 2.5 µg of antibody against H3K27ac (Abcam, ab4729) was added to sonicated chromatin of 2 × 106 cells and incubated overnight at 4 °C. Protein A sepharose beads (GE healthcare) were added to the ChIP reactions and incubated for 2 h at 4 °C. Beads were washed and chromatin was eluted. After crosslink reversal, RNase A and proteinase K treatment, DNA was extracted with the Monarch PCR & DNA Cleanup kit (NEB). Sequencing libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions. The quality of dsDNA libraries was analyzed using the High Sensitivity D1000 ScreenTape Kit (Agilent) and concentrations were assessed with the Qubit dsDNA HS Kit (Thermo Fisher Scientific). Libraries were single-end sequenced on a HiSeq 4000 (Illumina). ChIP-seq reads were aligned to the human reference genome build hg19 with bowtieEGA dataset EGAD00001007582

本研究针对一批入选患者开展了染色质免疫共沉淀测序（ChIP-seq）实验，以探究增强子（enhancer）与启动子（promoter）区域的表观遗传变化。ChIP实验操作参照既往文献并做小幅修改²⁷。简要流程如下：于室温下用1%甲醛将细胞交联10分钟，随后以终浓度0.125 M的甘氨酸（glycine）终止交联反应。采用Covaris S220聚焦超声破碎仪（Covaris S220 focused-ultrasonicator）对染色质进行超声破碎，使其平均片段长度达到250–350 bp。取2×10⁶个经超声破碎的染色质样本，加入2.5 μg靶向H3K27ac的抗体（货号ab4729，购自Abcam），于4℃条件下孵育过夜。向ChIP反应体系中加入Protein A琼脂糖磁珠（Protein A sepharose beads，GE healthcare），于4℃孵育2小时。随后对磁珠进行洗涤并洗脱结合的染色质。完成交联逆转后，经RNase A与蛋白酶K（proteinase K）处理，使用Monarch PCR与DNA纯化试剂盒（Monarch PCR & DNA Cleanup kit，NEB）提取DNA。按照制造商说明书，采用NEBNext Ultra II DNA文库制备试剂盒（NEBNext Ultra II DNA Library Prep Kit，适配Illumina平台，NEB）构建测序文库。使用高灵敏度D1000 ScreenTape试剂盒（High Sensitivity D1000 ScreenTape Kit，Agilent）对双链DNA文库的质量进行检测，采用Qubit dsDNA HS试剂盒（Qubit dsDNA HS Kit，赛默飞世尔科技，Thermo Fisher Scientific）定量文库浓度。最终在HiSeq 4000测序平台（Illumina）上对文库进行单端测序。测序reads使用bowtie比对至人类参考基因组hg19，相关数据集已存入EGA数据库，编号为EGAD00001007582。