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, 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.

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, 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.

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 Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c, 12-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age, sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight, ground controls and basal groups had blood collected, then were euthanized, had one hind limb removed, and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection.

The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c, 12-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age, sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight, ground controls and basal groups had blood collected, then were euthanized, had one hind limb removed, and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection.

The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c, 12-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age, sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight, ground controls and basal groups had blood collected, then were euthanized, had one hind limb removed, and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection.

The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c, 12-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age, and matching sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight, ground controls and basal groups had blood collected, then were euthanized, had one hind limb removed, and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection. Brain samples from three flight and three ground control animal groups were cut in half between hemispheres. One hemisphere of each brain was used for generating spatially resolved transcriptional profiling data. Hemispheres were cryosectioned so that 2 consecutive sections from the hippocampus of each brain was placed on Visium Gene Expression arrays. Samples were fixed, stained with Hematoxylin and Eosin and imaged. Imaging was followed by tissue permeabilization to release mRNA molecules from cells for capture onto the array surface. Subsequently, following the 10XGenomics Visium Gene Expression protocol, Spatial Transcriptomics RNA-seq libraries were prepared and sequenced. The other hemisphere of each brain was used for single nuclei RNA-seq and ATAC-seq using the 10X Multiome protocol. In addition, bulk RNA-seq (ribodepleted, target depth of 60 M clusters, PE 150 bp) was performed from a pool of RNA extracted from 10-20 sections from each of 3 flight and 3 ground control samples.

The objective of the Rodent Research-7 mission (RR-7) was to study the impact of the space environment on the gut microbiota of two strains of mice and how any changes in-turn affect the immune system, metabolic system, and circadian or daily rhythms. To this end, ten 11-week-old female C57BL/6J and ten 11-week-old female C3H/HeJ mice were flown to the International Space Station on June 29, 2018 on SpaceX-15 and housed in two Rodent Habitats. Samples of food, swabs from living surfaces, and fecal pellets were collected from each animal before launch and regularly during the mission. The mission also involved extended video collection (48 hr video segments per Habitat) to monitor circadian rhythms, and on-orbit mass measurement. After 25 days on-orbit, half of the mice of each strain were euthanized on the ISS with Ketamine/Xylazine/Acepromazine and cardiac puncture, after which carcasses were segmented in three sections and preserved in RNA later. After 75-76 days the remaining 5 animals from each group were euthanized and processed in the same manner. The 25-day dissected carcasses returned on SpX-15, and the 75-day dissected carcasses returned on SpX-16. 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 (same habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups, which were euthanized and processed on the same schedule and in the same manner as the flight samples. Dissections for tissues from all experimental groups were completed by the PI groups along with NASA's Biospecimen Sharing Program in February 2019. GeneLab received dorsal skin samples from forty C57BL/6J mice: 10 Basal, 5 Ground (25 days), 5 Ground (75 days), 5 Flight (25 days), 5 Flight (75 days), 5 Vivarium (25 days), 5 Vivarium (75 days). GeneLab received dorsal skin samples from forty C3H/HeJ mice: 10 Basal, 5 Ground (25 days), 5 Ground (75 days), 5 Flight (25 days), 5 Flight (75 days), 5 Vivarium (25 days), 5 Vivarium (75 days). From these skin samples, RNA was extracted, libraries generated (stranded, ribodepleted) and sequenced (target 60 M clusters at PE 98 bp).

In the Rodent Research Reference Mission (RRRM-1), forty female BALB/cAnNTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 10-12 week-old and twenty 32 week-old mice were flown, respectively. To directly assess spaceflight effects, half of the young and old mice (10 old, 10 young) were sacrificed on-orbit after 22-23 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 40 days and allowed to recover for 2 days (Live Animal Return, LAR) before sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice housed in flight hardware in matched environmental conditions), basal controls (10 mice sacrificed 1 day after launch), and vivarium controls (10 mice housed within standard vivarium habitats). Thus RRRM-1 included a total of 160 mice. This datasets features ribodepleted total RNA-seq data from livers dissected from all groups in RRRM-1. Data from 7-10 livers per group are included. All samples include either Mix 1 or Mix 2 of the ERCC spike-in control.

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 was 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). Fecal pellets were isolated directly from mice during dissection and preserved by flash freezing in liquid nitrogen before stored at -80 C. DNA was then extracted, shotgun metagenomic libraries generated, and libraries sequenced (target 10 M clusters at PE 250 bp). Metagenomic data was generated from the following groups: Basal Control (n=5), Ground Control (n=5), Vivarium Control (n=5), Cohort Control 1 (n=5), Cohort Control 2 (n=5), Flight (n=5).