GSM6938075: RNA_1g_laminin_2; Homo sapiens; RNA-Seq
Spaceflight samples, HEK293: Cells in a disposable cultivation chamber were thawed, washed with ice-cold PBS, lysed with 500 µl of lysis buffer and triturated ten times in a syringe with a 30-gauge needle. Spaceflight samples, C. elegans: Frozen nematode culture (1.5-4.5 ml) was thawed in 10 ml of wash buffer. The adult worms were filtrated through a 30-µm ÜberStrainer (pluriSelect Life Science) and washed three times with wash buffer. Then, the nematodes were pelleted by centrifugation at 1,600 × g for 1 min at 4°C, resuspended in 600 µl of lysis buffer, dripped into liquid nitrogen, and pulverized using the Multi-beads Shocker at 2800 rpm for 10 sec. Simulated microgravity samples: Cells in DCC were washed with PBS and lysed with 500 µl of lysis buffer. Mouse soleus muscles: Frozen soleus muscles were pulverized with 600 µl of frozen lysis buffer containing cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche) and 1000 U/ml RNase inhibitor, Murine (New England Biolabs [NEB]), using the Multi-beads Shocker at 3000 rpm for 30 sec for 2 cycles. For standard ribosome profiling, RNase I treatment, ribosome pelleting, size selection, preadenylated linker ligation, rRNA depeletion, reverse transcription, cDNA circularization, and PCR amplification. For Thor-Ribo-Seq, preadenylated linkers with the antisense sequence of T7 promoter were ligated and footprints were amplified by in vitro transcription. For RNA-Seq, the libraries were prepared by SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input (TaKaRa) or TruSeq Stranded mRNA Library Prep kit (Illumina).