Bulk RNA sequencing and spatially resolved transcriptional profiling of cerebellums from mice flown on the RR-10 mission

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.

啮齿类研究-10任务（Rodent Research-10，简称RR-10）的目标为探究太空飞行如何影响在轨正常骨组织再生的细胞与分子机制。为此，10只14~15周龄的雌性B6129SF2/J野生型（Wild Type，WT）小鼠，与10只14~15周龄的雌性B6;129S2-Cdkn1atm1Tyj/J（p21敲除，p21-null）小鼠，在飞行前接受了骨标记物茜素红（Alizarin Red）的皮下注射，随后通过SpaceX-21任务被运送至国际空间站（International Space Station，ISS）。在安乐死的7天前，全部20只小鼠均接受了骨形成标志物钙黄绿素（Calcein）的腹腔（intraperitoneal，IP）注射。在安乐死的48±2小时前，全部20只小鼠再次接受钙黄绿素的腹腔注射，并同时注射了细胞增殖标志物溴脱氧尿苷（BrdU）。在微重力环境中停留28~29天后，飞行组小鼠被实施安乐死。摘除后肢后，将躯体用铝箔包裹，置于低温冷藏箱（CryoChiller）中保存，并在-80℃及以下温度储存直至返回地球。除飞行组外，本研究还设置了3个地面对照组：基础对照组（Basal，代表发射前状态）、饲养笼对照组（Vivarium，在标准饲养笼中饲养时长与飞行组一致）以及地面模拟对照组（Ground，在国际空间站环境模拟器ISSES中使用飞行舱体进行饲养）。每个对照组每组均纳入20只小鼠（每种基因型各10只），仅饲养笼对照组每组每种基因型纳入12只。所有对照组小鼠均按照与飞行组完全一致的流程进行处理、安乐死及样本制备。本研究包含来自5只野生型飞行组小鼠与5只野生型地面对照组小鼠小脑的批量RNA测序及空间分辨转录谱分析数据。研究人员将右侧小脑半球进行包埋并冷冻切片，冷冻切片一部分用于批量RNA测序，另一部分置于基因表达芯片上，随后进行染色与成像。成像完成后对组织进行透化处理，以释放细胞内的mRNA分子，使其被捕获至芯片表面。后续制备空间转录组文库并进行测序。