The transcriptome of Acipenser schrenckii infected by Mycobacterium marinum (infection group)
Two groups of Acipenser schrenckii were infected with Mycobacterium marinum and mock-infected with PBS respectively. After 22d, when the fish in the infection group showed obvious symptoms, the livers of four fish in each group were collected for RNA-seq.Total RNA was extracted from the tissues with TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The RNA integrity value (RIN) was checked with a RNA 6000 Pico LabChip on an Agilent 2100 Bioanalyzer (Agilent, USA). The RNA samples were treated with Dnase I (Ambion,USA) under 37℃ for 1 hour to remove DNA residue, and mRNAs were purified using Micropoly (A) PuristTM mRNA purification kit (Ambion, USA) according to the manufacturer’s instructions. The concentration of mRNA was determined by NanoDrop (Thermo Scientific, USA). The RNA samples from each group were pooled before library construction. Double-stranded cDNA was synthesized following the previous protocol15. In brief, first-strand cDNA synthesis was primed by a GsuI-oligodT primer, 10 mg of mRNA, and 1000 units of Superscript II reverse transcriptase (Invitrogen) when incubation at 42℃ for 1 hr. Thereafter,  the mRNA’s CAP structure was oxidized by NaIO4 (Sigma) and ligated to biotin hydrazide, which was used to link complete mRNA/cDNA hybrids to Dynabeads M-280 (Invitrogen) for purification. The mRNA in hybrids were removed by alkaline treatment. The 5’-end specific adaptors were added to synthesize the second strand of cDNA. Then the poly(A) tails were removed by GsuI digestion. cDNAs were then fragmented into length of 300 ~ 500 bp, after purification with Ampure beads (Agencourt,USA), sequencing adaptors were ligated to the fragments.The cDNA libraries were constructed with Truseq DNA sample Prep kit (Illumina,USA), subsequent sequencing run was performed on illumina platform.