RNA_Seq of Phaeodactylum tricornutum
Total RNA was extracted by Tiangen Magnetic Bead Method Plant Polysaccharide Polyphenol Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Beijing), the concentration and purity of RNA were detected by NanoDrop 2000(Thermo Scientific), and the special agarose electrophoresis for RNA was used (electrophoresis apparatus: DYY-6C, Beijing June 1st; Gel imaging system: GelDoc 2000,BIO-RAD; Agarose:111860,Biowest) was used to test its integrity. total RNA with total amount > =1ug was selected. Use nebnext ultra ii RNA library prep kit for Illumina, Enriching mRNA with polyA tail by Oligo(dT) magnetic beads, randomly interrupting the mRNA with divalent cations, synthesizing cDNA with fragmented mRNA as template and random oligonucleotide as primer, purifying double-stranded cDNA, then carrying out double-end repair, introducing "A" base at 3' end and connecting sequencing linker, The cDNA of 400-500 bp was screened by AMPure XP beads, and the PCR products were amplified by PCR. Finally, the library was obtained by using Agilent 2100 Bioanalyzer (Agilent, 2100). The quality of the library was detected by Agilent High Sensitivity DNA Kit (Agilent, 5067-4626), and the total concentration of the library was detected by Pico green (Quanti Fluor-ST Fluorometer, Promega, E6090; Quant-Itpico Green DSDNA Assay Kit, Invitrogen, P7598), QCPR quantitative detection of effective library concentration (Thermoscientific step one plus real-time PCR systems).