Female C57BL/6 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. Purified RNA samples that were used for microarray analysis for GLDS-25 were provided to GeneLab. GeneLab added ERCC control spike-in to the samples and performed RNA-Seq analysis.

Mice were flown onboard STS-135 and returned to Earth for analysis. Livers were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Samples were shipped to UCI Genomics High Throughput Facility for analysis.

Mice were flown onboard STS-135 and returned to Earth for analysis. Livers were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Samples were shipped to UCI Genomics High Throughput Facility for analysis.

Mice were flown onboard STS-135 and returned to Earth for analysis. Livers were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Samples were shipped to UCI Genomics High Throughput Facility for analysis.

Mice were flown onboard STS-135 and returned to Earth for analysis. Livers were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Samples were shipped to Metabolon Inc. for analysis.

Mice were flown onboard STS-135 and returned to Earth for analysis. Cerebellums were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Samples were shipped to UCI Genomics High Throughput Facility for analysis.

The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective Beta-2 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated or implanted with vehicle or treatment-filled nDS launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return [LAR]) or >50 days (N=20 ISS Terminal). After 29 days the 20 LAR animals were returned live to back to Earth on January 13 2018,. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Baseline groups of animals sacrificed (LAR Baseline & FLT Baseline; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). A Ground Control group mimicked the Flight LAR group which was housed at KSC then shipped alive to Novartis Facilities where both the LAR and LAR Ground Control groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS Terminal mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR and Baseline. Following blood draw and hind limb dissection the ISS-terminal animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80C or colder until return. The ISS-terminal Ground Controls (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-terminal frozen ISS-terminal Ground Controls and frozen 0-day FLT baseline animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received samples of liver from only sham treated animals (no drug treated animals) from the following groups Flight: LAR (n=10) ISS Terminal (n= 10); Ground Controls: LAR GC (N=9) ISS Terminal GC (N=10) LAR Baseline (n=10) ISS Terminal Baseline (n=10). Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150).

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).

To understand the molecular mechanisms affected by spaceflight it is essential to achieve high quality sample preservation on-orbit for downstream gene expression analysis. However sample preservation protocols must also be compatible with available equipment and crew time. NASA s Rodent Research (RR) missions have used various methods for euthanasia carcass preservation and tissue preservation. This study extends the sample preservation study performed by GeneLab in GLDS-49 which examined conditions used for the RR-1 mission to include conditions used for multiple RR missions and is designed to help determine factors which may confound data analysis. To determine whether these various factors affect changes in gene expression this ground-based study generated gene expression profiles measured by RNAseq from the livers of 20-21 week-old female C57BL/6J mice. Multiple interacting factors were investigated: 1) To understand how euthanasia protocols affect gene expression when mouse carcasses are slow frozen mice were euthanized by either euthasol injection ketamine/xylazine injection or CO2 inhalation and carcasses slow frozen on dry-ice mimicking carcass preservation in the MELFI on the ISS. Carcasses were thawed and RNA extracted from livers; 2) To understand how carcass preservation protocols affect gene expression mice were euthanized with euthasol and carcasses preserved by flash freezing in liquid nitrogen slow freezing on dry ice or immersion in RNAlater following three-way segmentation. Carcasses were thawed and RNA extracted from livers; 3) To understand how tissue preservation protocols affect gene expression mice were euthanized with euthasol and livers dissected and processed immediately or preserved by flash freezing in liquid nitrogen slow freezing on dry ice or immersion in RNAlater. Liver samples that were processed immediately were homogenized in RLT buffer and then either immediately further processed for RNA extraction or were stored for 70 days at -80C post-homogenization in sample RLT buffer prior to RNA extraction.

The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective Beta-2 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated or implanted with vehicle or treatment-filled nDS launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return [LAR]) or >50 days (N=20 ISS Terminal). After 29 days the 20 LAR animals were returned live to back to Earth on January 13 2018,. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Baseline groups of animals sacrificed (LAR Baseline & FLT Baseline; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). A Ground Control group mimicked the Flight LAR group which was housed at KSC then shipped alive to Novartis Facilities where both the LAR and LAR Ground Control groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS Terminal mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR and Baseline. Following blood draw and hind limb dissection the ISS-terminal animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80C or colder until return. The ISS-terminal Ground Controls (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-terminal frozen ISS-terminal Ground Controls and frozen 0-day FLT baseline animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received samples of colon from only sham treated animals (no drug treated animals) from the following groups Flight: LAR (n=10) ISS Terminal (n= 9); Ground Controls: LAR GC (N=8) ISS Terminal GC (N=9) LAR Baseline (n=9) ISS Terminal Baseline (n=9). Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150).