library_construction_protocol: Novogene uses standard extraction methods to extract RNA from tissues or cells, and then strictly controls the RNA samples, and the quality control standards mainly include the following three aspects. 1.Agarose gelelectrophoresis: Samples are analyzed for RNA integrity and the presence of DNA contamination. 2.NanoPhotometer spectrophotometer：Detection of RNA purity（OD260/280and OD260/230）。 3.Agilent 2100 bioanalyzer：Accurate detection of RNA integrity. Note: RNA integrity and total volume were used as the main reference indicators There are two main ways to obtain mRNA: one is to use the structural feature that most of the mRNA in eukaryotes has polyA tails, and enrich mRNAs with polyA tails through Oligo(dT) magnetic beads. The second is to remove ribosomal RNA from total RNA to obtain mRNA. Subsequently, the mRNA obtained was randomly interrupted with divalent cations in the NEB Fragmentation Buffer, and the library was created according to the normal NEB library preparation method or the strand-specific library preparation method.  1.NEB common library construction: The first cDNA strand was synthesized in M-MuLV reverse transcriptase system using fragmented mRNA as template and random oligonucleotides as primer, then the RNA strand was degraded by RNaseH, and the second cDNA strand was synthesized using dNTPs as raw material in DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tail was added, and sequencing joints were connected. cDNA of about 250-300bp was screened by AMPure XP beads for PCR amplification, and PCR products were purified by AMPure XP beads again, and finally A library was obtained. The NEBNext® Ultra™ RNA Library Prep Kit was used for Illumina®.2.Chain specific database: The method for synthesizing the first strand of cDNA by reverse transcription is the same as the common library building method of NEB. The difference is that when synthesizing the second strand, dTTP in dNTPs is replaced by dUTP, and then the cDNA end repair, A-tail addition, sequencing joint connection and length screening are also carried out. Then, USER enzyme was used to degrade the second strand of cDNA containing U, and PCR amplification was performed to obtain the library. Chain specific library has many advantages, such as the same amount of data can obtain more effective information; More accurate gene quantitative, localization and annotation information can be obtained. It provides antisense transcripts and a single exon expression level in each isoform. The NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® was used. NEB common library construction: The first cDNA strand was synthesized in M-MuLV reverse transcriptase system using fragmented mRNA as template and random oligonucleotides as primer, then the RNA strand was degraded by RNaseH, and the second cDNA strand was synthesized using dNTPs as raw material in DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tail was added, and sequencing joints were connected. cDNA of about 250-300bp was screened by AMPure XP beads for PCR amplification, and PCR products were purified by AMPure XP beads again, and finally A library was obtained. The NEBNext® Ultra™ RNA Library Prep Kit was used for Illumina®. Chain specific database: The method for synthesizing the first strand of cDNA by reverse transcription is the same as the common library building method of NEB. The difference is that when synthesizing the second strand, dTTP in dNTPs is replaced by dUTP, and then the cDNA end repair, A-tail addition, sequencing joint connection and length screening are also carried out. Then, USER enzyme was used to degrade the second strand of cDNA containing U, and PCR amplification was performed to obtain the library. Chain specific library has many advantages, such as the same amount of data can obtain more effective information; More accurate gene quantitative, localization and annotation information can be obtained. It provides antisense transcripts and a single exon expression level in each isoform. The NEBNext® Ultra™ Directional RNA Library Prep Kit was used for Illumina