Time-course microarray transcriptome data of in vitro cultured testes and age-matched in vivo testes

In vitro spermatogenesis, which produces fertile spermatozoa, has been successfully performed using an organ culture method from murine tissue. Here, we provide a dataset of time-course microarray transcriptome data of in vitro cultured neonate murine testes and age-matched in vivo-derived testes. The dataset presented here is related to the article titled “Transcriptome analysis reveals inadequate spermatogenesis and immediate radical immune reactions during organ culture in vitro spermatogenesis” published in Biochemical and Biophysical Research Communications in 2020 [1]. The raw data and pre-processed data are publicly available on the GEO repository (accession number GSE147982). Furthermore, the dataset provided here includes additional metadata, detailed explanations of the experiment, results of pre-processing, analysis scripts, and lists of differentially expressed genes from in vitro culture testes and in vivo testes at each time point.


a b s t r a c t
In vitro spermatogenesis, which produces fertile spermatozoa, has been successfully performed using an organ culture method from murine tissue. Here, we provide a dataset of time-course microarray transcriptome data of in vitro cultured neonate murine testes and age-matched in vivo -derived testes. The dataset presented here is related to the article titled "Transcriptome analysis reveals inadequate spermatogenesis and immediate radical immune reactions during organ culture in vitro spermatogenesis" published in Biochemical and Biophysical Research Communications in 2020 [1] . The raw data and pre-processed data are publicly available on the GEO repository (accession number GSE147982). Furthermore, the dataset provided here includes additional metadata, detailed explanations of the experiment, results of pre-processing, analysis scripts, and lists of differentially ex-

Value of the Data
• This is the first time-course transcriptome data of in vitro cultured testes, which is useful for describing the difference between in vitro cultured testes and in vivo testes. • The data benefits scientists in reproductive medicine and reproductive engineering, especially those involved in in vitro spermatogenesis. • The data can be used to identify target pathways/genes to improve in vitro spermatogenesis.

Data Description
RNA was extracted from in vitro cultured testes (2, 4, 6, 7, 9, and 14 days of culture from 7 dpp mouse testes) and age-matched control in vivo -derived samples (7, 9, 11, 13, 14, 16, and 21 dpp mouse testes), followed by microarray analyses in three biological replicates. A schematic representation of the data generation is shown in Fig. 1 . The raw intensity binary and text data of Illumina MouseWG-6 v2.0 Expression beadchip are provided as supplementary files (GSE147982_RAW.tar and GSE147982_non-normalized.txt.gz files, respectively) at Gene Expression Omnibus (GEO) ( https://www.ncbi.nlm.nih.gov/geo/ ) with the accession number GSE147982. The link between each GEO sample accession and the original mouse is provided in Table 1 . The preprocessed data, which is subjected to background correction, variance-stabilizing transformation (VST), and quantile normalization, are also provided at GEO GSE147982. The distributions of signal intensity of raw and preprocessing data are reported in Fig. 2 . Supplementary Table 1 provides the result of differential expression analysis, which is done using an empirical moderated t-statistics test with a cut-off adjusted p-value of 0.05.

Organ culture
Testes were extracted from 7 dpp male Acr-Gfp transgenic mice. The extracted testes were decapsulated and cut into 4-8 fragments by forceps under a microscope in the culture medium Table 1 Origin of RNA sample. The abbreviation "dpp" corresponds days post-partum (days-old) and "cddp" corresponds corresponding days post-partum, which is age-matched sample to the in vivo derived testes. Rep means biological replicate index of an experiment. ( α-minimum essential medium (Thermo Fisher Scientific Inc., Wilmington, NC, USA)), supplemented with 40 mg/mL AlbuMAX (Thermo Fisher Scientific Inc.). The testis fragments were then patterned onto a 1.5% (w/v) agarose gel block (about 10 mmW × 10 mmD × 5 mmH) that was pre-soaked for at least two days in advance and then half-submerged in the culture medium in a well of a 12 well tissue-culture plate (Greiner Bio-One, Kremsmünster, Austria). The testes fragments were covered with the Polydimethylsiloxane (PDMS)-ceiling chip [3] and cultured in 5% CO 2 at 34 °C with once a week medium replacement.

RNA extraction
Fresh in vivo -derived testes were maintained in RNA later (Thermo Fisher Scientific Inc.) on ice for at least 3 h until RNA extraction. The in vivo-derived testes and in vitro cultured testes were homogenized in TRIzol Reagent (Thermo Fisher Scientific Inc.) with the PT1300D polytron homogenizer (KINEMATICA AG, Luzern, Switzerland), and the aqueous phase was collected according to the manufacturer's instructions. RNA was extracted from the corrected aqueous phase using the NucleoSpin RNA (MACHEREY-NAGEL, Düren, Germany), according to the manufacturer's instructions. The quality of the RNA was confirmed by NanoDrop (ND-10 0 0, Thermo Fisher Scientific Inc.) and RNA 60 0 0 nano kit of Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples with high RNA integrity values ( > 9) were used in the microarray analyses. The upper panel is raw intensity (before pre-processing) and the lower panel is after pre-processing. The abbreviation "dpp" corresponds days post-partum (days-old) and "cddp" corresponds corresponding days post-partum, which is age-matched sample to the in vivo derived testes. Rep means biological replicate index of an experiment.

Microarray
Total RNA was amplified by in vitro transcription method and the resulting cRNAs were biotinylated using the Illumina TotalPrep RNA Amplification Kit (Illumina, Inc., San Diego, CA, USA). The biotinylated cRNA was hybridized to MouseWG-6 v2.0 Expression beadchip (Illumina, Inc.). Signal intensity was measured using the Illumina BeadArray reader (Illumina, Inc.).

Data pre-processing
Raw intensity binary data (idat files) were converted to text data using BeadStudio (I llumina, Inc.) without background correction and normalization. Raw intensity text data were subjected to background correction, variance-stabilizing transformation (VST), and quantile normalization using lumiExpresso function implemented in the lumi package of R.

Differential expression analysis
The probes whose detection p-value was greater than 0.01 in any of the samples were removed. A linear model was fitted to the expression data for each probe using the lmFit function implemented in the limma package of R. Then, p-values of empirical Bayes moderated t-statistics test between in vitro cultured-and in vivo -derived-testes at each time point were computed by contrasts.fit and eBayes functions implemented in the limma package of R, followed by p-values adjustment by Benjamin -Hochberg method. The probes were considered to be differentially expressed genes if their adjusted p-value was less than 0.05.

Scripts
Scripts and data for R analysis used in this paper are available on GitHub (https://github.com/RIKEN-CFCT/ivs_data_in_brief).

Declaration of Competing Interest
The authors have no conflicts of interest to declare.

Ethics Statement
All animal experiments conformed to the ARRIVE guidelines and the National Institutes of Health Guide for the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Animal Research Center of Yokohama City University, Yokohama, Japan)(Approval number: 17-A-17-072).