Female C57BL/6CR mice were flown onboard STS-135 for 13 days and returned to Earth for analysis. Livers were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Liver tissue samples that were used for microarray analysis for GLDS-25 were provided to GeneLab. GeneLab extracted RNA added ERCC control spike-in to the samples and performed RNA-Seq analysis.

The objective of the Rodent Research-9 (RR-9) mission was to use mice to understand the molecular basis of phenomena that affect astronauts during long-duration spaceflight particularly visual impairment and joint tissue degradation. To this end a flight group (FLT) of 10 week-old male C57BL/6J mice were launched from Kennedy Space Center (KSC) on 8/14/2017 and housed in Rodent Habitats on the ISS for 33 days before being returned alive to Earth. After splashdown in the Pacific Ocean the animals were transported to Loma Linda University (LLU) for testing euthanasia and dissection on 9/18/2018. A Basal Control (BSL) was housed in standard cages at Kennedy Space Center (KSC) and euthanized one day after launch of the FLT animals (8/15/2017). Ground Control (GC) and Vivarium Control (VIV) studies were planned to commence at KSC approximately one-week after the conclusion of the flight experiments. However all the GC and VIV mouse studies at KSC had to be cancelled due to Hurricane Irma and potential adverse effects on the animal housing facility. The GC and VIV studies were therefore rescheduled and begun in May 2018. The GC was euthanized and dissected 6/18/2018 - 6/20/2018 while the VIV was euthanized and dissected 6/22/2018 - 6/23/2018. Because this resulted in a different cohort of mice being used for the GC and VIV controls as compared to the flight (FLT) and basal (BSL) groups two cohort controls were included in the study. The first Cohort Control 1 (CC_C1) was from the same cohort as the FLT and BSL animals and was sacrificed and dissected 4 days after the FLT group (9/22/2017). The second Cohort Control 2 (CC_C2) was from the same cohort as the GC and VIV animals and was sacrificed and dissected 2-8 days after the GC and VIV groups (6/24/2018 - 6/26/2018). The CC_C1 and CC_C2 groups were housed in standard cages and fed standard chow in contrast to all other groups which received Rodent Foodbars. To clarify the connections between treatment groups and animal cohorts the following group abbreviations are used in the sample metadata: Flight (FLT_C1); Basal (BSL_C1); Ground Control (GC_C2); Vivarium Control (VIV_C2) Cohort Control 1 (CC_C1); Cohort Control 2 (CC_C2). Upon dissection livers were preserved in liquid nitrogen and stored at -80 C before RNA was extracted libraries generated (stranded ribodepleted) and sequenced (target 60 M clusters at PE 150 bp).

Data from the NASA Rodent Research-1 (RR-1) mission showed that gene-expression levels in mouse livers are different depending on what tissue preservation protocol is used and that slow freezing is not an effective method for preserving signals in gene-expression data. In response to these and other observations the Rapid Freeze hardware was built for use on the International Space Station. The Rapid Freeze hardware freezes mouse tissues (Glovebox freezer) and whole carcasses (Cryochiller) at rates closely mimicking those attained with immersion in liquid nitrogen. Because this hardware will be used extensively on future rodent research missions it is crucial to understand whether or not it preserves signals in gene expression data in order to maximize the value of these rare and expensive spaceflight experiments. Therefore this study was designed with three goals: 1) To evaluate the temperature profile of the Cryochiller and Glovebox freezer cartridges (Rapid Freeze hardware) over time during mock on-orbit procedures; 2) To determine the freezing profiles of tissues and carcasses using Rapid Freeze hardware at both optimal and sub-optimal temperatures (to mimic on-orbit operations) compared with those frozen in liquid nitrogen (the laboratory gold standard) or frozen at -80 C (the current standard method); 3) To identify gene expression changes in a) tissues that were frozen via the Glovebox freezer and b) tissues dissected from whole or partial carcasses that were frozen via the Cryochiller versus tissues that were frozen via control methods (liquid nitrogen or -80C slow freeze) to assess how the Rapid Freeze hardware compares with laboratory gold standard practices and our current standard methods.

The objective of the Rodent Research-10 mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. This study includes bulk RNA sequencing ribodepleted gene expression data from 10 Basal animals (5 WT and 5 p21-null), 9 Flight animals (4 WT and 5 p21-null), 10 Ground animals (5 WT and 5 p21-null), and 10 Vivarium animals (5 WT and 5 p21-null).

RR-1 is a validation flight to evaluate the hardware operational and science capabilities of the Rodent Research Project on the ISS. RNA DNA and protein were purified from liver tissues from RR-1 mice (female C57BL/6J 16wk old at time of launch). From each group two liver samples were collected and frozen immediately after euthanasia. The rest of the liver samples from each group were collected from frozen carcasses dissected post-flight. RNA-Seq whole genome and RNA BS-Seq (bisulfite sequencing) and proteomic expression profiling were performed. RNA extracted from these tissues was re-sequenced; these data are available as part of GLDS-168 (https://genelab-data.ndc.nasa.gov/genelab/accession/GLDS-168).

NASA's Rodent Research (RR) project is playing a critical role in advancing biomedical research on the physiological effects of space environments. Due to the limited resources for conducting biological experiments aboard the International Space Station (ISS), it is imperative to use crew time efficiently while maximizing high-quality science return. NASA's GeneLab project has as its primary objectives to 1) further increase the value of these experiments using a multi-omics, systems biology-based approach, and 2) disseminate these data without restrictions to the scientific community. The current investigation assessed viability of RNA, DNA, and protein extracted from archived RR-1 tissue samples for epigenomic, transcriptomic, and proteomic assays. During the first RR spaceflight experiment (RR-1), a variety of tissue types were harvested from subjects, snap-frozen or RNAlater-preserved, and then stored at least a year at -80C after return to Earth. They were then prioritized for this investigation based on likelihood of significant scientific value for spaceflight research. All tissues were made available to GeneLab through the bio-specimen sharing program managed by the Ames Life Science Data Archive and included mouse adrenal glands, quadriceps, gastrocnemius, tibialis anterior, extensor digitorum longus, soleus, eye, and kidney. We report here protocols for and results of these tissue extractions, and thus, the feasibility and value of these kinds of omics analyses. In addition to providing additional opportunities for investigation of spaceflight effects on the mouse transcriptome and proteome in new kinds of tissues, our results may also be of value to program managers for the prioritization of ISS crew time for rodent research activities.

The objective of the Rodent Research-23 missions (RR-23) was to better understand the effects of spaceflight on the eyes, specifically on the structure and function of the arteries, veins, and lymphatic vessels that are needed to maintain vision. To this end, twenty male, C57BL/6J, 16-17 weeks old mice were delivered to the ISS on SpaceX-21 in a single transporter, transferred to two rodent habitats, and maintained in microgravity for 38 days. Flight mice were then returned to Earth alive (Jan 13th, 2021). After splashdown in the Atlantic Ocean, mice were transported to Kennedy Space Center via helicopter. The 20 Flight, 20 Habitat Ground Control (HGC), and 20 Vivarium Ground Control (VGC) mice were removed from Rodent Transporters (Flight and HGC) or vivarium cages (VGC), placed into shipping containers, and flown to Texas A and M University. There, mice underwent post flight procedures, before euthanasia and tissue collection. Flight, HGC and VGC animals were euthanized and dissected on Jan 14th, 17th or 20th of 2021, respectively. Spleens were preserved by immersion in RNAlater and stored at -80C until RNA was extracted, and libraries generated and sequenced (target 60 M clusters per sample, PE 150 bp). This dataset features 9 samples from the Flight group, 10 samples from the HGC group, and 9 samples from the VGC group.

The objective of the Rodent Research-10 mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. This study includes bulk RNA sequencing data from left cerebral hemispheres from 4 WT flight animals and 5 WT ground control animals, and single nuclei transcriptomics and epigenomics data from left cerebral hemispheres from 5 WT flight animals, and 5 WT ground control animals.

The objective of the Rodent Research-10 mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. This study includes bulk RNA sequencing and spatially resolved transcriptional profiling data from cerebellums from 5 WT flight animals, and 5 WT ground control animals. Cerebellums from the right hemisphere were embedded and cryosectioned. Cryosections were either processed for bulk RNA sequencing or placed on gene expression arrays, stained and imaged. Imaging was followed by tissue permeabilization to release mRNA molecules from cells for capture onto the array surface. Subsequently, spatial transcriptomics libraries were prepared and sequenced.

This study was under the umbrella of the NASA Rodent Research-1 project consortium. In this project, 16-week-old female C57 BL/6J mice were sent to International Space Station (ISS) in NASA's animal enclosure modules (AEM) and were kept inside ISS middeck for 37 days. We used transcrptomic and methylomic approaches to investigated the spaceflight-associated neuro-ocular syndrome (SANS) using retinas from those spaceflight mice.