GSM5135217: DS1-PRJ-HCMV-22; Homo sapiens; ncRNA-Seq
The growth media from infected MRC-5 cells were spun on SW28 rotor (Beckman) at 80,000 x g for 1 hour at 4°C. The supernatant was discarded and the pellet in each tube was resuspended in 50 µl of cold phosphate buffered saline (PBS). About 0.7 ml of combined pellet suspension from all ultracentrifuge tubes was loaded on 15%-50% linear gradient of sucrose made in PBS and spun in SW41 (Beckman) rotor at 80,000 x g for 1 hour at 4°C. About 1 ml of visible virus-containing band was collected into a new tube and mixed with 10 ml of sterile PBS solution in order to dilute sucrose. The virions were concentrated at 80,000 x g for 1 hour at 4°C and resuspended in 0.1 ml of PBS and used for total RNA isolation. 3 µg of total RNA isolated from RNase pre-treated virions was used for snoRNA library preparation. Total RNA of each sample was used to prepare the SnoRNA-sequencing library in the following steps: 1) 3'-adapter ligation; 2) 5'-adapter ligation; 3) cDNA synthesis; 4) PCR amplification; 5) size selection of ~170-370bp PCR amplified fragments (corresponding to ~50-250 nt small RNAs). The libraries are qualified and quantified using Agilent BioAnalyzer2100.