GSM7813991: △J/△J-like; Deinococcus radiodurans; RNA-Seq
Wild-type and mutant cells were cultured until reaching an OD600 of 0.8. They were then centrifuged and resuspended in PBS buffer at pH 7.0. Total RNA was extracted from two independent biological replicates for each group using the TRIzol method following the manufacturer's protocol (Ambion, Foster City, CA, United States). RNA quality, including degradation and contamination, was assessed by electrophoresis on 1% agarose gels. Additionally, RNA integrity was evaluated using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The transcriptome Library preparation, clustering and sequencing were performed by Novogene Corporation (Beijing, China). Two micrograms of RNA per sample was used as the input material for library preparation, and then, a Ribo-ZeroTM Magnetic Kit (Epibio, MRZB12424) was used to remove rRNA. Subsequently, the obtained mRNA is randomly fragmented using divalent cations in the Fragmentation Buffer. Using the fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA is synthesized in the presence of MMuLV reverse transcriptase (NEB, USA). Then, the RNA strand is degraded with RNase H (NEB, USA), and the second strand of cDNA is synthesized using dUTP instead of dTTP as the raw material in the presence of DNA polymerase I. The purified double-stranded cDNA undergoes end repair, adenylation, and adapter ligation. USER enzyme (NEB, USA) is then added to degrade the second cDNA strand containing uracil (U). The cDNA fragments of approximately 370-420 bp are size-selected using AMPure XP beads, followed by PCR amplification. The PCR products are purified again using AMPure XP beads to obtain the final library.