Jan 09, 2026
RNA sequencing (RNA-seq) and analysis
- Fanxuan Tian1
- 1Department of Gastroenterology, Chongqing Key Laboratory of Digestive Malignancies, Daping Hospital, Chongqing 400042, China
Protocol Citation: Fanxuan Tian 2026. RNA sequencing (RNA-seq) and analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv55jkjv1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 08, 2026
Last Modified: January 09, 2026
Protocol Integer ID: 238277
Keywords: data analysis for cultured murine cell, cultured murine cell, analysis rna, rna, sequencing, cell, seq
Abstract
RNA sequencing and data analysis for cultured murine cells, such as B16F10.
Troubleshooting
Total RNA is extracted from cultured cells using TRIzol reagent (15596018CN, Invitrogen).
Total RNA is quantified using a NanoDrop spectrophotometer and an Agilent 2100 bioanalyzer (Thermo Fisher).
RNA library construction and subsequent RNA sequencing are performed using BGI-Shenzhen (Shenzhen, China) software.
First-strand cDNA is generated by random hexamer-primed reverse transcription followed by second-strand cDNA synthesis.
A-Tailing Mix and RNA Index Adapters are added by incubation for end repair.
The cDNA fragments obtained from the previous step are amplified by PCR and the products are purified using Ampure XP Beads and dissolved in an EB solution.
The product is validated using an Agilent Technologies 2100 Bioanalyzer for quality control.
Double-stranded PCR products from the previous step are denatured and circularized using splint oligo sequences to obtain the final library.
Single-stranded circular DNA is used as the final library. The final library is amplified with phi29 to form a DNA nanoball, which has over 300 copies of one molecule and is loaded into the patterned nanoarray, and single end 100 bases reads are generated on the BGIseq500 platform (BGI-Shenzhen).
SOAPnuke (v1.5.6)
Citation
LINK
is used to filter out adapter sequences and eliminate poor-quality bases from raw sequencing data. A read is excluded if its sequence closely matched the adapter sequences.
Reads are excluded if they contained > 20% low-quality bases (base quality < 15) or > 5% unknown bases.
Clean reads are mapped to Ensemble GRCm38 using HISAT.
Citation
LINK
To obtain gene expression data, RNA-Seq by Expectation Maximization (RSEM)
Citation
LINK
is used to count the number of genes and quantify gene expression by calculating the Fragments Per Kilobase of exon per Million mapped reads (FPKM) for downstream analysis.
Citations
Step 10
Chen Y, Chen Y, Shi C, Huang Z, Zhang Y, Li S, Li Y, Ye J, Yu C, Li Z, Zhang X, Wang J, Yang H, Fang L, Chen Q. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data.
https://doi.org/10.1093/gigascience/gix120Step 12
Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements.
https://doi.org/10.1038/nmeth.3317Step 13
Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.
https://doi.org/10.1186/1471-2105-12-323