Novogene RNA quality assessment and library-preparation methods
Received: 2026-09-10

Source documents:
File: methods.pdf
Page: 1 (printed page 0)
SHA256: b7342abec6dac2c5acb733862df6843dfb4595076c6633b22b570244b82e4b31

File: methods_Chinese.pdf
Page: 1 (printed page 0)
SHA256: 571e26a4b6a593a117f6de92bb3e98c7adbe87ff9fb24e016d669de89fa67dd1

Scope: RNA quality assessment and library preparation. Project sample counts, differential-expression results and software versions are documented separately in parameter_records.tsv using the original six-sample report.
The RNA Nano 6000 Assay Kit is recorded for RNA integrity assessment.

methods.pdf, page 1: extracted original text (PDF line breaks and spacing retained)
RNA quantification and qualification 
RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the  Bioanalyzer 
2100 system (Agilent Technologies, CA, USA). 
Library preparation for Transcriptome sequencing 
Total RNA was used as input material for the RNA sample preparations.  
Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. 
Fragmentation was carried out using divalent cations under elevated  temperature in First 
Strand Synthesis Re action Buffer(5X). First strand  cDNA was synthesized using random 
hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. 
Second strand cD NA synthesis was subsequently  performed using DNA Polymerase I and 
dNTP. Remaining overhangs were  converted into blunt ends via exonuclease/polymerase 
activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure 
were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 
370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman 
Coulter, Beverly, USA). Then PCR was performed  with Phusion High -Fidelity DNA  
polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified 
(AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

methods_Chinese.pdf, page 1: extracted original text (PDF line breaks and spacing retained)
1、RNA 提取与检测 
Agilent 2100 bioanalyzer：精确检测RNA完整性和总量。 
2、文库构建与质检 
建库起始RNA为total RNA，总量>=1ug。通过Oligo(dT)磁珠富集带有polyA 尾的
mRNA，随后在Fragmentation Buffer中用二价阳离子将得到的 mRNA 随机打断。以片
段化的mRNA 为模版，随机寡核苷酸为引物，在M-MuLV 逆转录酶体系中合成cDNA 
第一条链，随后用RNaseH 降解RNA 链，并在DNA polymerase I 体系下，以dNTPs 为
原料合成cDNA 第二条链。纯化后的双链 cDNA经过末端修复、加A 尾并连接测序接
头，用 AMPure XP beads 筛选370~420 bp 左右的 cDNA，进行 PCR 扩增并再次使用
AMPure XP beads 纯化PCR 产物，最终获得文库。文库构建完成后，先使用 Qubit2.0 
Fluorometer 进行初步定量，稀释文库至1.5ng/ul，随后使用Agilent 2100 bioanalyzer 对
文库的insert size 进行检测，insert size 符合预期后，qRT-PCR 对文库有效浓度进行准
确定量（文库有效浓度高于2nM），以保证文库质量。
