Data of RNA-seq transcriptomes of liver, bone, heart, kidney and blood in klotho mice at a pre-symptomatic state and the effect of a traditional Japanese multi-herbal medicine, juzentaihoto

We performed RNA-seq analyses of mRNA isolated from five organs, liver, bone, heart, kidney and blood at the pre-symptomatic state of klotho mice with/without administration of a Japanese traditional herbal medicine, juzentaihoto (JTT). Data of differentially expressed genes (DEG) with/without JTT was included. Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. We collected data in which retained-introns were accumulated in a particular set of genes of these organs, and showed that among these retained introns in the liver and bone a subset was recovered to the normal state by the medicine. All of the data present changes of molecular events on the levels of metabolites, proteins and gene expressions observed at the pre- symptomatic state of aging in klotho mice with/without JTT. The research article related to this Data in Brief is published in GENE entitled as “Intron retention as a new pre-symptomatic marker of aging and its recovery to the normal state by a traditional Japanese herbal medicine”.

in the liver and bone a subset was recovered to the normal state by the medicine. All of the data present changes of molecular events on the levels of metabolites, proteins and gene expressions observed at the pre-symptomatic state of aging in klotho mice with/without JTT. The research article related to this Data in Brief is published in GENE entitled as "Intron retention as a new pre-symptomatic marker of aging and its recovery to the normal state by a traditional Japanese herbal medicine".  Parameters for data collection Male α-klotho knockout mice and wild-type mice were bred from 3.5 weeks after birth in a JTT-administered group and a non-administered group. At 7 weeks, mouse tissues were removed from each group and RNA was extracted. Sequencing library construction from mRNA was prepared according to the supplier's manual. Sequencing was performed the supplier's manual for paired-end sequencing. Description of data collection Mouse tissues were removed from each group and RNA was extracted using Pure Link RNA Mini kit (Invitrogen). Sequencing library construction for Illumina NovaSeq 60 0 0 from mRNA was prepared according to the supplier's manual using

Value of the Data
• The data provides that retained-introns accumulated in the pre-symptomatic state, and a subset of them was changed into the administration of traditional Japanese herbal medicine. • The data provides proof-of-concept evidence related to the ancient Chinese statement proposing the medicine's usefulness for treating the pre-symptomatic state. Accordingly, it encourages all the researchers who are trying to get molecular evidence in the field of herbal medicines. • The data propose that stress-dependent intron retention is a biomarker for pre-symptomatic and aging. This proposal is presented only using a limited set of organs, mainly klotho mice.
To strengthen the generality, it is necessary to use many different or ganisms and their organs, including humans. This data will provide a basis for future research in the search for biomarkers of stress response and aging.

Data Description
In relation to the related research article, we collected the data which represented the presymptomatic state (7 weeks of age) of klotho mice and the recovery from this state to the healthy state on the molecular level by administration of traditional Japanese herbal medicine, juzentaihoto (JTT) [1][2][3] . Western blot analysis showed that the pattern of protein expressions at this state is not so much changed from that of wild type ( Fig. 1 ). Metabolome analysis showed that the state is under a starvation-like condition ( Fig. 2 ). RNA was isolated from five organs (liver, blood, kidney, heart and bone) and their transcriptomes were determined by using RNAseq. Integrity of RNA-seq data was confirmed by MDS analysis ( Fig. 3 ). Analyses of differentially expressed genes in the blood between klotho mice with JTT (KL + ) and those without JTT (KL-) showed that JTT upregulated genes related to mitochondria including oxidative phosphorylation ( Fig. 4 ). Similarly, genes involved in heme biosynthesis were upregulated by administration of JTT in the blood of KL + ( Fig. 5 ). By using rMATS software ( Fig. 6 A), we showed that retained introns were accumulated even at the pre-symptomatic state, a subset of which were recovered to the healthy state by JTT in the liver. In the case of wild type, administration of JTT does not change the gene expression profile in WT + with that of WT-( Fig. 6 B & 6 C). Fig. 7 shows that JTT can completely recover the 13 intron-retained loci in the bone to the healthy state by administration of JTT. Fig. 8 shows their own characteristics of IR loci in the case of genes from the bone, in terms of intron length, GC% of intron sequences and the strength score of 5 / 3 śplice sites. Fig. 9 shows that a very few TFs may control genes with increased intron (IncIR) in liver, blood and bone.

Experimental design
α-klotho knockout ( Kl −/ − Jcl, klotho mice) mice have the phenotype of human progeria, with a lifespan of approximately 10 weeks, and are a model mouse for aging studies. Klotho mice ( N = 10) and wild-type (WT, N = 10) 3-week-old male mice were purchased from CLEA Japan. Each mouse was acclimated for 3 days in a vinyl isolator at the breeding facility in CLEA Japan. Then, klotho mice and WT mice were bred separately in a group fed with juzentaihoto (JTT)containing feed and a normal diet group, respectively. One group was fed CE-2 containing 0.5% (w/w) JTT powder and the other group was fed CE-2 without JTT. JTT is a traditional Japanese herbal medicine, the powdered extract of which is supplied by Tsumura (Tokyo, Japan), and is made by spray-drying boiling extracts from a mixture of 10 crude medicines. JTT contains 10.52% Astragali radix, 10.52% Cinnamomi cortex, 10.52% Rehmanniae radix, 10.52% Paeoniae radix, 10.52% Cnidii rhizome, 10.52% Atractylodis lanceae Rhizome, 10.52% Angelicae Ginseng, and 5.32% Glycyrrhizae radix. CE-2 is a general feed for rodents that contains vegetable protein as the main ingredient, as well as animal protein, fat, fiber, carbohydrates, vitamins, and minerals. Mice in each group were dissected at 7 weeks of age according to the animal ethics regulations, and liver (3 in each group), heart (4 in each group), bone (5 in each group), and kidney (5 in each group) were removed; those for RNA sequencing were soaked in RNALater and stored in the freezer, and a portion of liver tissue was frozen intact for metabolomic analysis. Whole blood (0.8 mL) was collected from the tail vein using a syringe containing 8-12 μL of EDTA-2 K (4 in each group). Then, equal volumes of PBS buffer and 3 vol of TRIzol (Thermo Fisher Scientific) were added, dispensed into tubes, and stored frozen. The experimental procedures using animals were approved by the Laboratory Animal Committee of CLEA Japan. Fig. 1 )
The homogenate solutions were centrifuged at 10,0 0 0 × g for 20 min at 4 °C. The protein concentration of the supernatant was measured with a Poerce BCA protein assay kit (Thermo       Fisher Scientific). The supernatant was diluted to equalize the protein concentrations, and DTT (FUJIFILM Wako) was added to make the final concentration of 0.2 M. Then, 4 × SDS sample buffer (6% SDS, 40% glycerol, 0.4% bromophenol blue, 250 mM Tris, pH 6.8) was added and boiled at 95 °C for 5 min. The prepared protein samples (30 μg/well) were separated by electrophoresis on SDS-PAGE (10% gel) for 1-2 h at 20 mA/gel. After electrophoresis, they were transferred onto PVDF membranes (GE Healthcare) using wet transfer for 2 h at 100 mA on ice. The membranes were blocked with 5% skim milk in TBST (20 mM Tris, 150 mM NaCl, containing 0.05% Tween-20, pH 7.4), blotted with the antibodies described below. Primary antibodies used in this study were anti-DDX5, p68 (  Boxplots showing intron lengths, GC% of intron sequences, and the strength score of 5 / 3' splice sites compared among three groups of introns, namely "no recovery", "complete recovery" and "All introns" (254,005 loci) in the bone. Statistical analyses are unpaired Student's t tests, and significance is annotated as * : P < 0.05, * * : P < 0.01, * * * : P < 0.001.
we used the secondary antibodies peroxidase-conjugated anti −rabbit IgG (SA0 0 0 01-2, Proteintech) 1:16,0 0 0 or anti-mouse IgG (sc-516,102, Santa Cruz Biotechnology) 1:10,0 0 0 in TBST for 1 h at room temperature. After incubation, the membranes were washed three times in TBST for 10 min each, and protein bands were detected using enhanced chemiluminescence (ECL) Prime Western Blotting Detection Reagents (GE Healthcare). One of the membranes was probed with anti-GAPDH, and used as the loading control for other blots in each experiment. The signal intensity was quantified using ImageJ software (NIH, Bethesda, MD) or a ChemiDoc system (Bio-Rad). Western blots were repeated at least three times with different animals, and representative blots are shown.

Measurement of metabolites (related to Fig. 2 )
Metabolome measurements and data processing were carried out through a facility service at Human Metabolome Technologies Inc., Tsuruoka, Japan. 50 mg of frozen liver tissue was crushed with 1500 μL of 50% acetonitrile/Milli-Q water containing an internal standard (Solution ID: 304-1002, Human Metabolome Technologies, Inc.) at 0 °C in a tissue homogenizer (Micro Smash MS100R, Tomy Digital Biology Co., Ltd., Tokyo, Japan). The homogenate was centrifuged at 2300 × g at 4 °C for 5 min. Then, 800 μL of the upper aqueous layer was filtered through a Millipore filter (5-kDa cutoff) at 9100 × g at 4 °C for 120 min to remove proteins. The filtrated solution was concentrated by centrifugation and resuspended in 50 μL of Milli-Q water.
Metabolome analysis was performed by capillary electrophoresis −time-of-flight mass spectrometry (CE-TOFMS) analysis. CE-TOFMAS experiments were performed using Agilent CE-TOFMAS system (Agilent Technologies Inc., Santa Clara, CA, USA). Separations were performed with the aid of a capillary filled with fused silica (50 mm internal diameter × 80 cm total length) filled with 1 M formic acid or 50 mM ammonium acetate (pH 8.5) as the electrolyte for cation or anion analyses, respectively. The applied voltage for separation was set at 30 kV. MS spectra were scanned from m/z 50 to 10 0 0. MS was conducted in negative ionization mode with 3500 V for anion analysis and in positive ionization mode with 40 0 0 V for cation analysis. Data processing for peak picking, peak alignment, metabolite annotation, and peak integration was performed using MasterHands TM (ver.2.17.1.11)(Institute for Advanced Biosciences, Keio University, JAPAN). Fig. 3 ) Total RNA was extracted using the Pure Link RNA Mini kit (Invitrogen, MA, USA) according to the manufacture's protocol. Briefly, 600 μL of lysis buffer and 900 μL of TRIzol (Thermo Fisher Scientific) were added to 30 mg of individual tissue samples (liver, heart and kidney), and homogenized. After incubation at room temperature for 10 min, the samples were centrifuged at 12,0 0 0 × g for 15 min, the supernatant was vortexed with 200 μL of 1-Bromo-3-chloropropane (BCP) and centrifuged at 12,0 0 0 × g for 15 min. For blood samples, 200 μL of BCP was added per 1.5 mL of each blood samples (with PBS buffer and TRIzol), vortexed, and centrifuged at 12,0 0 0 × g for 15 min. The supernatant was treated with an equal volume of 70% ethanol and was purified by using column cartridge. During the washing procedure, the sample was treated with DNase (Invitrogen) on the column. After washing, RNA was eluted with 50-100 μL of RNase-Free Water. To confirm RNA quality, RNA concentration was measured using qubit (Thermo Fisher Scientific) and RIN values were confirmed using TapeStation (Agilent Technologies, CA, USA). For RNA-seq, RIN values of 6 or higher were used. RNA library was constructed by using a TruSeq Stranded mRNA Sample Prep kit (Illumina, CA, USA). Paired-end sequencing (150 b × 2) was outsourced to Takara Bio (Shiga, Japan) using a NovaSeq 60 0 0 system (Illumina).

RNA extraction, RNA-seq, quality check, filtering of RNA-seq data and mapping analysis (related to
Quality filtering was used to examine the RNA sequences obtained from the next generation sequencers. First, the adapter sequences for Illumina that were ligate to prepare the sequencing library using cutadpt software v.1.16 were trimmed. (Optional parameters: -m 30 -b GATCGGAA-GAGCACACGTCTGAACTCCAGTCAC -b AGATCGGAAGAGCGTCGTGTAGGGAAAGTGT) [4] . The poly(A) sequence was then trimmed using the fastx_clipper software supplied with the fastx toolkit software package v.0.0.14 [5] . were trimmed and short sequences that were less than 30 bases after trimming were removed using fastq_quality_trimmer software (Optional parameters: -t 20 -l 30 -Q 33) and fastq_quality_filter software (Optional parameters: -q 20 -p 80 -Q 33) included in the fastx toolkit software package. In the previous process, reads with one of the pairs missing were removed using Trimomatic v.0.38 software (Optinal parameters: MINLEN:30) [6] . Next, reads containing mouse rRNA and phiX standard sequences were detected and removed using Bowtie 2 v.2.3.4.1 software (parameters: default) [7] . The removal of unpaired reads was then performed again using bam2fastq software [8] . After these quality filtering, 20 million reads each of forward and reverse sequences per sample were mapped to the mouse genome sequence build GRCm38 using Tophat v2.1.1 (parameters: default) [9] . Mouse genome sequences were downloaded from the iGenomes of Illumina website [10] . Multiple mapping reads were then removed from the output BAM files using samtools software (Optional parameter: samtools view -q 4) [11] . Uniquely mapped reads by gene annotation (Ensembl release 81) were counted using FeatureCounts v.1.6.2 software [12] . The counted raw RNA expression values were normalized by the Trimmed mean of M values (TMM) method [13] using the EdgeR library [14] in R v.3.5.0, and used for expression analysis.
2.6. Splice site score (related Fig. 8 ) To evaluate splice site intensity at intron retention loci, MaxEntScan (parameters: default) [17] was used to calculate maximum entropy scores for 5 ánd 3 śplice sites. Fig. 9 ) Protein binding motifs in mRNA were compared to RNA binding proteins and their associated motifs in the ATtRACT database [18] . Transcription factor binding sites were detected using HOMER software [19] . All detected motifs were compared by the Fisher's test.

Ethics Statement
Experimental procedures using animals were carried out with approval from the Laboratory Animal Committee of CLEA Japan.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
No data was used for the research described in the article.