北京博奥晶典生物技术有限公司 第 1 页 共 6 页 illumina 转录组测序简明实验流程 一、实验基本流程图 Sample Preparation & QC End Repair & A-Tailing & Adapter Ligation PCR Enrichment Library QC Cluster Generation Sequencing by Synthesis mRNA Library Construction PolyA-mRNA purification&Fragment 2nd Strand cDNA Synthesis 1st Strand cDNA Synthesis 北京博奥晶典生物技术有限公司 第 2 页 共 6 页 二、mRNA 建库流程 1. 材料准备 1.1. 主要试剂 Code Reagents Manufacturer Cat No. 1 NEBNext Ultra RNA Library Prep Kit for Illumina NEB E7530S 2 NEBNext Poly(A) mRNA Magnetic Isolation Module NEB E7490S 3 NEBNext Multiplex Oligos for Illumina(Index Primers Set1) NEB E7335S 4 NEBNext Multiplex Oligos for Illumina(Index Primers Set2) NEB E7500S 5 RNA 6000 Pico chip Agilent 5067-1524 6 High Sensitivity DNA Assay Kit Agilent 5067-4626 7 RNA 6000 Nano chip Agilent 5067-1511 8 QUBIT RNA BR ASSAY KIT Invitrogen Q10211 9 QUBIT DNA BR ASSAY KIT Invitrogen Q32855 10 QUBIT DNA HS ASSAY KIT Invitrogen Q32854 11 KAPA SYBR FAST Master Mix Universal 2X qPCR Master Mix KAPA Biosystem KK4602 12 DNA Quantification Standards and Primer Premix Kit KAPA Biosystem KK4808 1.2. 主要仪器设备 Code Instruments Manufacturer Facility NO. 1 96-well thermal cycler (with heated lid) MJ B01-02 2 Bioanaly z er 2100 Agilent J06-02 3 QUBIT 2.0 FLUOROMETER Invitrogen Q32871 4 Peltier Thermal Cycler PTC-225 MJ B01-01 1.3. 主要耗材 耗材 型号 生产厂家 一次性吸头 0.5-10 L, 2-200 L,100-1000 L Ax y gen 一次性薄壁离心管 0.2 mL, 1.5 mL Ax y gen Gel Breaker Tubes 3388-100 IST Engineering 5 μm filter tube 5388-50 IST Engineering 北京博奥晶典生物技术有限公司 第 3 页 共 6 页 2. 样品准备和 QC 选择质量合格的 Total RNA 作为 mRNA 测序的建库起始样品,其质量要求通过 Agilent ...