RNA sequencing data for gamma radiation response in the extremotolerant tardigrade Ramazzottius varieornatus

Tardigrades are microscopic animals of which terrestrial species are capable of tolerating extreme environments by entering a desiccated ametabolic state known as anhydrobiosis. Intriguingly, they survive high dosage gamma rays (>4,000 Gy), possibly through a mechanism known as cross-tolerance. We hypothesized that anhydrobiosis genes are also regulated during cross-tolerance, thus we submitted Ramazzottius varieornatus to 500 Gy 60Co gamma-ray and conducted time-course low-input RNA-Seq. The gene expression was quantified with RSEM and differential expression was determined with DEseq2. Differentially expressed genes were submitted to gene ontology enrichment analysis with GOStat. The transcriptome dynamically shifted nine hours post-exposure.


a b s t r a c t
Tardigrades are microscopic animals of which terrestrial species are capable of tolerating extreme environments by entering a desiccated ametabolic state known as anhydrobiosis. Intriguingly, they survive high dosage gamma rays ( > 4,0 0 0 Gy), possibly through a mechanism known as crosstolerance. We hypothesized that anhydrobiosis genes are also regulated during cross-tolerance, thus we submitted Ramazzottius varieornatus to 500 Gy 60 Co gamma-ray and conducted time-course low-input RNA-Seq. The gene expression was quantified with RSEM and differential expression was determined with DEseq2. Differentially expressed genes were submitted to gene ontology enrichment analysis with GO-Stat. The transcriptome dynamically shifted nine hours postexposure.

Value of the Data
• These data are the first preliminary data of valuable time-course transcriptome sequence data of the extremotolerant tardigrade, Ramazzottius varieornatus , exposed to a high dosage of 60 Co gamma-ray (500 Gy), providing the community a basis for studying the gamma-ray response in tardigrades. • These data provide researchers new insights about molecular processes affected after gamma-ray exposure in tardigrades and the expression of genes with unknown functions. • The data shown here would be a basis of understanding the anti-oxidative stress response in tardigrades, leading to the identification of anhydrobiosis related factors.

Data Description
This manuscript presents a time-course transcriptomic dataset obtained from the gamma-ray exposure-response of the extremotolerant tardigrade, Ramazzottius varieornatus . This RNA-Seq data employed low-input RNA-Seq, 30 ng of total RNA extracted from 30 individuals per sample. Table 1 displays all RNA-Seq data obtained in this study. The gene expression was quantified with RSEM based on the bowtie2 mapping against the coding sequences of R. varieornatus ( Table S1 ), and a total of 4,138 genes was determined differentially expressed by DESeq2 (FDR Table 1 Summary of RNA-Seq data and mapping ratio to coding sequences and whole genome. < 0.05, Figs. 1 , S1 , Tables S1-S3 ). Gene ontology enrichment analysis of between all-time points illustrates the transcriptomic response against the extensive stress caused by the gamma-ray exposure ( Table S4 ). Using this data requires caution, as sampling was conducted in technical triplicates and may show less variance that may be seen between biological replicates. TMM-normalized TPM values of differentially expressed genes were Z-scaled and visualized as a heat map. The spearman correlation between each gene were clustered into 8 clusters with the Ward method, indicated by the numbers on the right end of the plot. Ctrl: Non-irradiated controls, h04-h24: samples from 4h-24h post exposure.

Low-input time-course RNA-Seq
Tardigrades were reared based on the method established by our previous study [1] . R. varieornatus were placed on 90 mm plastic plates layered with 2% volvic agar at 22 °C and fed with Chlorella vulgaris . Culture plates were transported to Japan Atomic Energy Agency (JAEA), Takasaki Advanced Radiation Research Institute, and were incubated at 22 ˚C for more than 12 hours to eliminate the effects of the transportation. 2,0 0 0 tardigrades were suspended in two 1.5 mL tubes and filled with 100 μL of distilled water. These tubes were then irradiated with 500 Gy of 60 Co gamma ray, at a dose rate of 2,008 Gy/hour at a distance of 63 cm from the 60 Co source. The content of each tubes was spread over a 90 mm agar layered plate and incubated at 22 °C until further sampling. At each sampling point (4h, 6h. 9h, 12h, 15h, 18h, 24h), 30 animals were randomly sampled with 10 μL Pipettes with the least water as possible, and placed into 200 μL tubes containing 150 μL of TRIzol (Life Technologies, 3 technical replicates per condition), and were preserved at −20 °C. Total RNA was extracted with Direct-Zol (Zymo Research) and 20 ng of total RNA was submitted to cDNA synthesis, sequencing adaptor and index ligation, PCR amplification using NEBNext Ultra RNA Kit (New England BioLabs Japan) according to manufacture protocol. RNA was extracted at least within 1 week of irradiation. The cDNA was amplified by at least 15 cycles of PCR (h1-1~h6-2: 15 cycles, h6-3~h21-3: 21 cycles, h24-1~h24-3: 23 cycles). The quality of the nucleic acids was validated with Nanodrop (Thermo Scientific) and quantified by Qubit RNA High Sensitivity or dsDNA Broad Range (Life Technologies). The library length distribution was validated by Tapestation D10 0 0 (Agilent). Since sample h09_1 showed signs of adapter dimers, the pooled samples were size selected for the main band. Sequencing was performed with NextSeq 500 (Illumina) following manufacturer's instructions, using NextSeq 500 High Output Kit (75bp single end). Raw reads were de-multiplexed by bcl2fastq v2.15.0.4 (Illumina) and each sequencing lane were merged. Each sample were validated with FastQC v0.11.2 [2] .

Informatics analysis
Gene expression was quantified with RSEM using the align_and_estimate_abundance.pl utility from the Trinity suite v2.11.0 [3] using the R. varieornatus coding sequence [4] . Reads were also mapped to the genome with bwa MEM v0.7.12-r1039. The raw mapped counts were tested for differential expression with DESeq2 v1.22.2 [5] and genes with FDR < 0.05 and fold change above 2 were called as differentially expressed genes. Gene expression profiles were clustered by Ward method based on the spearman's correlation and was visualized as a heatmap with ggplot2. A gene ontology enrichment analysis was performed for each time point with GOstat v2.48.0 [6] , and terms with a single observation was excluded. G-language Genome Analysis Environment v1.9.1 [7 , 8] were used for sequence manipulation and data parsing.

Ethics Statement
All experiments were conducted following the Japanese law and guidelines from The Science Council of Japan, as well as the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.