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).

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 RR-23 mission 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. Livers were preserved by flash freezing in liquid nitrogen and stored at -80 C 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 and VGC groups, and 8 samples from the HGC group.

Harsh environmental conditions including microgravity and radiation during prolonged spaceflights are known to alter hepatic metabolism. Our studies have focused on the analysis of possible changes in metabolic pathways in livers of mice which experienced 30 days of spaceflight with and without an additional re-adaption period of 7 days compared to control mice on Earth. Utilizing shotgun mass spectrometry and label-free quantification we performed proteomic profiling to investigate mice livers from the spaceflight project xe2 x80 x9cBion-M 1 xe2 x80 x9d. No significant alterations in protein levels were observed between control mice liver and spaceflight mice which is possibly caused by insufficient fold change detection combined with high variances within the groups. In contrast our results show that more than a third of the quantified protein levels are altered comparing the liver proteome of mice with and without re-adaption time after their spaceflight. Proteins related to amino acid metabolism showed higher levels after re-adaption which may indicate higher rates of gluconeogenesis. Members of the peroxisome proliferator-activated receptor pathway reconstitute their level after 7 days due to a decrease in fold change which indicates decreased signs of non-alcoholic fatty liver disease. Moreover bile acid secretion regenerates on Earth due to reconstitution of related transmembrane proteins and elevated levels of the drug-metabolising enzymes belonging to the CYP superfamily decrease 7 days after the spaceflight. Thus our study demonstrates reconstitution of pharmacological response and early signs of non-alcoholic fatty liver disease recover within 7 days whereas glucose uptake should be monitored due to alterations in gluconeogenesis.

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).

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).

In the Rodent Research Reference Mission (RRRM-2), forty female C57BL/6NTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 12 week-old and twenty 29 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 55-58 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 32 days and allowed to recover for 24 days (Live Animal Return, LAR) before sacrifice. ISS-T and LAR mice were the same age at sacrifice. Both the ISS-T and LAR animals had independent ground controls (GC; 10 mice per group housed in flight hardware in matched environmental conditions), basal controls (BSL; 10 mice per group sacrificed 2 days before launch), and vivarium controls (VIV; 10 mice per group housed within standard vivarium habitats). Thus RRRM-2 included a total of 160 mice. This study includes bulk RNA sequencing ribodepleted gene expression data from hearts from 5 old and 5 young animals from each of the following groups: LAR basal, LAR ground control, LAR vivarium control, LAR flight, ISS-T basal, ISS-T ground control, and ISS-T flight. The ISS-T vivarium control young group has only 3 animals due to quality control issues.

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).

In the Rodent Research Reference Mission (RRRM-2), forty female C57BL/6NTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 12 week-old and twenty 29 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 55-58 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 32 days and allowed to recover for 24 days (Live Animal Return, LAR) before sacrifice. ISS-T and LAR mice were the same age at sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice per group housed in flight hardware in matched environmental conditions), basal controls (10 mice per group sacrificed 2 days before launch), and vivarium controls (10 mice per group housed within standard vivarium habitats). Thus RRRM-2 included a total of 160 mice. This study includes bulk RNA sequencing and single nuclei transcriptomics and epigenomics from left cerebral hemispheres from 4 young ISS-T spaceflight animals, 5 old ISS-T spaceflight animals, 5 young ISS-T ground control animals, and 4 young ISS-T ground control animals.

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).