Genome-wide maps of H3K36me3 in clear cell renal cell carcinoma (ccRCC) [ChIP-seq]

To address the need to study frozen clinical specimens using next-generation RNA, DNA, chromatin immunoprecipitation (ChIP) sequencing and protein analyses, we developed a biobank work flow to prospectively collect biospecimens from patients with renal cell carcinoma (RCC). We describe our standard operating procedures and work flow to annotate pathologic results and clinical outcomes. We report quality control outcomes, nucleic acid yields of our RCC submissions (N=16) to The Cancer Genome Atlas (TCGA) project, as well as newer discovery platforms by describing mass spectrometry analysis of albumin oxidation in plasma and 6 ChIP sequencing libraries generated from nephrectomy specimens after histone H3 lysine 36 trimethylation (H3K36me3) immunoprecipitation. From June 1, 2010, through January 1, 2013, we enrolled 328 patients with RCC. Our mean (SD) TCGA RNA integrity numbers (RINs) were 8.1 (0.8) for papillary RCC, with a 12.5% overall rate of sample disqualification for RIN <7. Banked plasma had significantly less albumin oxidation (by mass spectrometry analysis) than plasma kept at 25°C (P<.001). For ChIP sequencing, the FastQC score for average read quality was at least 30 for 91-95% of paired-end reads. In parallel, we analyzed frozen tissue by RNA sequencing and after genome alignments, only 0.2-0.4% of total reads failed the default quality check steps of Bowtie2, which was comparable to the disqualification ratio (0.1%) of the 786-O RCC cell line, prepared under optimal RNA isolation conditions. The overall correlation coefficients for gene expression between the Mayo Clinic vs. TCGA tissues ranged from 0.75 to 0.82. These data support the generation of high-quality nucleic acids for genomic analyses from banked RCC. Importantly, the protocol does not interfere with routine clinical care. Collections over defined time points during disease treatment further enhance collaborative efforts to integrate genomic information with outcomes. Overall design: Examination of H3K36me3 in ccRCC

为满足利用下一代RNA、DNA、染色质免疫沉淀（chromatin immunoprecipitation, ChIP）测序及蛋白质分析手段研究冷冻临床标本的需求，我们开发了一套生物样本库工作流程，用于前瞻性收集肾细胞癌（renal cell carcinoma, RCC）患者的生物标本。我们详述了用于标注病理结果与临床结局的标准操作流程及工作方案。本研究报告了提交至癌症基因组图谱（The Cancer Genome Atlas, TCGA）项目的16份肾细胞癌样本的质控结果与核酸产出情况，同时通过描述血浆白蛋白氧化的质谱分析结果，以及针对经组蛋白H3赖氨酸36三甲基化（histone H3 lysine 36 trimethylation, H3K36me3）免疫沉淀的肾切除术标本构建的6个ChIP测序文库，介绍了最新的发现型研究平台。2010年6月1日至2013年1月1日期间，我们共纳入328名肾细胞癌患者。我们测得的乳头状肾细胞癌样本的TCGA RNA完整性数值（RNA integrity number, RIN）均值（标准差）为8.1（0.8），其中RIN<7的样本整体不合格率为12.5%。相较于在25℃条件下保存的血浆，冻存血浆的白蛋白氧化水平显著更低（质谱分析结果，P<0.001）。针对ChIP测序，91%~95%的双端读段（paired-end reads）的FastQC平均读段质量评分不低于30。与此同时，我们对冷冻组织进行了RNA测序；经基因组比对后，仅0.2%~0.4%的总读段未通过Bowtie2的默认质控步骤，这一比例与在最优RNA分离条件下制备的786-O肾细胞癌细胞系的不合格率（0.1%）相当。梅奥诊所（Mayo Clinic）与TCGA组织样本间的基因表达整体相关系数介于0.75至0.82之间。上述数据证实，从冻存的肾细胞癌样本中可获取高质量核酸用于基因组分析。重要的是，本研究方案不会干扰常规临床诊疗流程。在疾病治疗期间的特定时间点进行标本采集，可进一步推动将基因组信息与临床结局相结合的合作研究。整体实验设计：检测透明细胞肾细胞癌（clear cell renal cell carcinoma, ccRCC）中的H3K36me3。