GSM8284498: Soleus skeletal muscle, subject 5, after 21-day dry immersion; Homo sapiens; RNA-Seq
Using the method of open-incision muscle biopsy under local anesthesia, samples of the soleus muscle were taken from all subjects 10-20 days before the start of the experiment (pre-immersion samples) and on the day of the end of the experiment (post-immersion samples). Soleus muscle samples were immediately frozen in liquid nitrogen and then stored in a low-temperature freezer at -80 °C. Total RNA was isolated from muscle tissue using the RNA isolation kit RNeasy Micro (Qiagen, Germany). 5 μg of purified total RNA from samples corresponding to 4 subjects (4 pre-DI samples and 4 post-DI samples) were concentrated in 5 μL of nuclease free water, using vacuum concentrator SpeedVac Savant SC210A (ThermoFisher Scientific, USA). Then this total RNA was used as a template for the synthesis of the first cDNA strand (nAnTiCAGE Library Preparation kit, DNAform, Japan and SuperScript III Reverse Transcriptase, Invitrogen, USA), which was then biotinylated at the 5' end of using (nAnTiCAGE Library Preparation kit, DNAform, Japan). This allowed selection of 5' cap contained RNAs using streptavidin beads (Dynabeads M270 Streptavidin, ThermoFisher Scientific, USA). At this stage, rRNA, truncated or not fully transcribed RNA was eliminated. For more efficient removal of nonspecific RNA strands, cDNA was treated by RNase I and H (nAnTiCAGE Library Preparation kit, DNAform, Japan) and then was purified using Agencourt AMPure XP (Beckman Coulter, USA). Next, linkers at the 5'and 3' ends (nAnTiCAGE Library Preparation kit, DNAform, Japan) were sequentially ligated to the cap-trapped cDNA. The 5' linker contains the XmaJI restriction endonuclease recognition and the MmeI class II restriction enzyme recognition site, as well as a barcode for multiplexing (nAnTiCAGE Library Preparation kit, DNAform, Japan). The 3' linker contains an XbaI restriction enzyme recognition site. After treatment with restriction enzymes, short CAGE tags were obtained (nAnTiCAGE Library Preparation kit, DNAform, Japan). At the final stage, a second cDNA strand was synthesized (nAnTiCAGE Library Preparation kit, DNAform, Japan). The concentration of the libraries was validated using qPCR (KAPA Library Quantification Kits Illumina, KAPA Biosystems, South Africa) and sequenced on a HiSeq 2500 platform (Illumina, USA) using the HiSeq version 4 reagent kit (HiSeq SR Cluster Kit version 4 cBot and HiSeq SBS Kit version 4 50 cycles, Illumina, USA) in the 60 bp single end mode.