Transcriptome Sequencing of Tumor-educated Lung Epithelial Cells in the Pre-metastatic Niche. Transcriptome Sequencing of Tumor-educated Lung Epithelial Cells in the Pre-metastatic Niche

We performed transcriptome sequencing on epithelial cells, isolated from lungs of normal and tumor-bearing mice, to shed light on the function and phenotype changes of lung epithelial cells in the pre-metastatic niche. Overall design: Lung tissues from tumor-bearing mouse and normal mouse (as a control) were digested into single-cell suspensions and CD45- SftpC+ epithelial cells were purified by FACS, followed by transcriptome sequencing. All procedures were performed according to the standard manufacturer’s protocol. The total RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads (for eukaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments (about 200 bp). Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR amplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library. The library products are ready for sequencing via Illumina HiSeqTM 2000 or other sequencer when necessary.

本研究对从正常小鼠及荷瘤小鼠肺部分离得到的上皮细胞开展转录组测序，以阐明肺上皮细胞在转移前微环境（pre-metastatic niche）中的功能与表型变化。
实验设计概述：将荷瘤小鼠与作为对照的正常小鼠的肺部组织消化为单细胞悬液，通过荧光激活细胞分选术（Fluorescence Activated Cell Sorting, FACS）纯化得到CD45⁻、SftpC⁺的肺上皮细胞，随后进行转录组测序。所有实验步骤均严格遵循试剂盒厂商提供的标准操作规程执行。
总RNA样品首先经脱氧核糖核酸酶I（DNase I）处理，以降解潜在的DNA污染。随后采用寡聚(dT)磁珠（适配真核样本）富集mRNA。将mRNA与片段化缓冲液混合，打断为约200 bp的短片段。以随机六聚体引物反转录合成第一链cDNA，随后加入缓冲液、脱氧核糖核苷三磷酸（dNTPs）、核糖核酸酶H（RNase H）及DNA聚合酶I合成第二链cDNA。采用磁珠纯化获得双链cDNA，随后进行末端修复及3'端单核苷酸腺嘌呤（A）加尾反应。最后将测序接头连接至片段化产物两端，通过PCR扩增富集目标文库片段。在质量控制（QC）环节，采用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）与ABI StepOnePlus实时荧光定量PCR系统对样品文库进行质控与定量。待文库制备完成后，可根据需求通过Illumina HiSeq™ 2000或其他测序平台开展测序。