Illumina NovaSeq 6000 paired end sequencing; Simulated microgravity regulates the self-renewal and pluripotency of mouse and human embryonic stem cells - RNA-seq
Simulated microgravity regulates the self-renewal and pluripotency of mouse and human embryonic stem cells - RNA-seq
Total RNA was purified by RNeasy Minikit (Qiagen) according to the manufacturer's manual. RNA concentration was determined by Nanodrop, and 500 ng of total RNA was used for library construction using KAPA Stranded mRNA-Seq Kits. Sequencing was performed on Illumina HiSeq 2500 machines with 125 bp pair-end mode. ESCs dissociated with 0.05% of trypsin were centrifuged and re-suspended in ES medium without LIF, and then plated in 25 ml cell culture flask to induce the formation of EBs. Cultures were maintained for indicated days and incubated at 37 °C with 5% CO2. EBs at indicated time were collected for qPCR analysis.A Rotary Cell Culture System (RCCS) (SM-31, National Space Science Center, Chinese Academy of Sciences, China) was used in this study to simulate microgravity. Total RNA was extracted using the RNeasy Minikit (Qiagen) based on the manufacturer's instructions. Library was prepared using KAPA Stranded mRNA-Seq Kits based on the manufacturer's instructions.