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, 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, 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.

In Rodent Research Reference Mission-1 (RRRM-1), also known as RR-8, 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 mammary glands dissected from six LAR groups (young and old for flight, ground control, and vivarium control). Data from 5-10 livers per group are included. All samples include either Mix 1 or Mix 2 of the ERCC spike-in control. Target depth was 60 M clusters sequenced at PE 150 bp.

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).