Research On RNA-seq Library Construction For Small Quantity Of Cells

Next-generation sequencing technology is a milestone in the history of sequencing technology.This technology enables efficient deep sequencing of millions of DNA molecules,revolutionizing gene expression regulation and functional research.RNA-seq acts as an important tool for transcriptome,deepening our understanding of cell subpopulations and gene function,and promoting the discovery of differential gene expression,which is helpful to elucidate the mechanism of disease.A variety of RNA-Seq technologies have been developed,such as Drop-seq,MARS-seq,and CEL-seq.However,in clinical individualized medicine,it is often necessary to perform RNA-seq analysis on rare tissues or a small quantity of cell to help disease diagnosis and treatment.However,RNA-seq technology for a small quantity of cells still encounters the problem of scarce RNA and low efficiency in library construction.In the RNA-seq library construction process,the Tn5 transposase is often used to rapidly fragment the cDNA,and the sequencing adapters can be ligated to both ends of the fragment to construct the library,shortening the library construction process,but the previous study found that the Tn5 transposase used in the process of RNA-seq for a small amount of cells,is easy to introduce a large number of ME(Mosaic End)sequences,which can generate non-specific amplification,forming a large number of invalid sequencing Reads.It reduces the quality of RNA-seq data and affects the analysis of subsequent sequencing results.In order to reduce the non-specific amplification of ME sequence in a small quantity of cell RNA-seq,we need to establish an efficient strategy for building RNA-seq library for a small quantity of cell.Objectives:1.To optimize the expression and purification of Tn5 transposase protein,improving its yield and purity and facilitating RNA-seq library construction for small quantity of cells;2.To establish a highly efficient RNA-seq library construction strategy for small quantity of cells and to reduce the nonspecific amplifications resulted from Tn5 ME sequence during RNA-seq library construction for small quantity of cells,leading to high efficiency in RNA-seq by Tn5 transposase.Methods:1.Tn5 protein coding gene was amplified from Tn5 plasmid by PCR and carried out homologous recombination with pET28a-6His-SUMO vector to get recombinant plasmid.The recombinant plasmid was transformed into Codon Plus BL21(DE3)to express Tn5 transposase after IPTG induction.Lysed by sonication,suspension was treated with DNase and RNase to remove DNA and RNA contamination.By NTA affinity purification,we purified Tn5 transposase.At last,elute Tn5 transposase protein from NTA beads by imidazole solution(high concentration)and analyze the protein products by SDS-PAGE.2.Synthesize Tn5ME-A,Tn5ME-B and Tn5-Rev sequence and anneal to yield double strand sequence.We carried out Tn5 transposase assembly with annealed sequence to get Tn5 transposome.To remove unbounded Tn5 ME sequence,which may lead to nonspecific amplification in the followed PCR,we transferred transposome to protein concentrator tube for purification,resulting in the removal of substances of which MW are under 30 KDa.Finally,we performed 1% agarose electrophoresis for products analysis.3.To detect the activity of purified Tn5 transposome,we tagmented CT DNA using purified Tn5 transposome and performed 1% agarose electrophoresis for products analysis.At the same time,we used RT-PCR to analyze products above in different concentrations with the conservative primers in Tn5 ME sequence.Finally,we compared the results with commercial Tn5 kit and used Tn5 transposome to construct RNA-seq library for small quantity of RAW264.7 cells.4.Sort 100 RAW264.7 cells by flow cytometry.After RNA extraction,we carried out reverse transcription and second strand cDNA synthesis to yield double strand cDNA.We constructed RNA-seq library for small quantity of RAW264.7 cells using purified Tn5 transposome and unpurified transposome respectively.Finally compare the differences between them.Results:1.The Tn5 protein coding gene and pET28a-6His-SUMO vector was successfully homologous recombined and the sequence was correct.Recombinant plasmid transformed into Codon Plus BL21(DE3)competent cells could successfully express Tn5 transposase protein.After sonication and purification by NTA,impure proteins in the lysate were removed by washing.Finally,Tn5 transposase protein was eluted by high concentration imidazole solution.SDS-PAGE results showed that the transposase protein of high purity could be obtained.2.Tn5 transposase protein was assembled with Tn5ME-A,Tn5ME-B and Tn5ME-Rev,forming a stable Tn5 transposome.After centrifuge with protein concentrator,the electrophoresis results of the purified products showed that the unbounded Tn5 ME sequence in the purified transposome was significantly reduced.3.The purified Tn5 transposome was used to tagment CT DNA.The electrophoresis results showed that the CT DNA of 6000 bp was tagmented,resulting in 500-1500 bp DNA smear.This indicated that CT DNA had been randomly tagmented to form DNA fragments smears.RT-PCR analysis of tagmentation products by transposome Tn5 at different concentrations showed that the sequencing adapters in transposome Tn5 were successfully inserted into DNA fragments,and the efficiency of Tn5 transposome diluted in 1:40 was as high as that of the commercial Tn5 kit.4.A small number of RAW264.7 cells were analyzed by RNA-seq with unpurified,purified and commercial Tn5 kit,and sequenced.Results showed that the purified Tn5 transposome could effectively reduce non-specific amplification,significantly avoiding invalid Reads,and found that the number of genes was twice that of the control(P < 0.05),greatly improving the quality of the RNA-seq library for small quantity of cells.Conclusions:1.The Tn5 transposome after expression and NTA purification reduced ME and impure protein contamination.2.The Tn5 transposome after assembly was purified by ultrafiltration using protein concentrator,which could remove Tn5 ME sequence.The nonspecific amplification of ME sequence in Tn5 transposome could be effectively reduced,and the library efficiency of Tn5 transposase in small quantity of cells could be dramatically improved.