Transcriptome Sequencing PPGL

RNA library preparation of samples with RNA Integrity Number (RIN) > 5.5 (median 7.1; range 5.6-8.9) was performed as described in the TruSeq Stranded mRNA Library Prep Kit (Illumina, RS-122-2101). Briefly, 0.7-1 µg total RNA PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters. Adapter-ligated library was completed by PCR with Illumina PE primers. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on an Illumina HiSeq2500 on a 51bp single-read format following manufacturer's protocols.
cDNA libraries from FFPE tumors and low integrity RNA’s (RIN<5.5) from FF tumors (median 2.4; range 1-5.5) but % of fragments > 200 nucleotides higher than 20% (median 57.5; range 20-96) were prepared using QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, 015) with a UMI Second Strand Synthesis Module for QuantSeq FWD (Lexogen, 081), vendor’s protocol for low input/ low quality/ FFPE RNA using 200-500 ng of total RNA, according to availability, was used as starting material. PCR Add-on Kit for Illumina (Lexogen, 020) was used to adjust the library amplification number of cycles. Libraries were applied to an Illumina flow cell for cluster generation and sequenced on NovaSeq6000.
Image analysis, per-cycle basecalling and quality score assignment was performed with Illumina Real Time Analysis software. Demultiplexing of BCL files to FASTQ format was performed with the bcl2fastq Software (Illumina).EGA study EGAS00001006044

RNA完整性指数（RNA Integrity Number, RIN）>5.5的样本（中位数7.1，范围5.6-8.9）的RNA文库制备，严格遵循TruSeq链特异性mRNA文库制备试剂盒（Illumina，货号RS-122-2101）的操作流程完成。简言之，取0.7-1 μg总RNA的PolyA+组分进行纯化与随机片段化，反转录为双链cDNA后，依次进行末端修复、dA尾加尾及接头连接等酶促处理；通过Illumina PE引物进行PCR扩增，完成接头连接后的文库构建。将纯化得到的cDNA文库加载至Illumina流动槽进行簇生成，随后依照厂商操作规范，在Illumina HiSeq2500平台上以51bp单端读长模式完成测序。福尔马林固定石蜡包埋（FFPE）肿瘤的cDNA文库，以及福尔马林固定（FF）肿瘤来源的低完整性RNA（RIN<5.5，中位数2.4，范围1-5.5）的cDNA文库，此类RNA中>200核苷酸的片段占比高于20%（中位数57.5%，范围20-96），采用QuantSeq 3’ mRNA-Seq文库制备正向试剂盒（Lexogen，货号015）搭配QuantSeq正向文库用UMI第二链合成模块（Lexogen，货号081）进行制备。以200-500 ng总RNA作为起始原料（用量根据样本实际可获取量调整），遵循厂商针对低起始量、低质量RNA及FFPE RNA的操作流程。使用Illumina PCR附加试剂盒（Lexogen，货号020）调整文库扩增的循环次数。将构建完成的文库加载至Illumina流动槽进行簇生成，随后在NovaSeq6000平台上完成测序。图像分析、每轮碱基识别与质量值赋值均通过Illumina Real Time Analysis软件完成。将BCL文件解多重并转换为FASTQ格式的操作，通过bcl2fastq软件（Illumina）完成。本数据集隶属于欧洲基因组档案（EGA）编号为EGAS00001006044的研究项目。