GSM5064877: COV014, small RNA; Homo sapiens; miRNA-Seq
The peripheral blood was collected into standard EDTA blood tubes and stored on ice until processed. The time of centrifugation should be as fast as possible. The upper plasma was separated after centrifuged at 2500 rpm with 20 minutes and frozen in the -80 refrigerator for standby procession. After all plasma samples were collected, the samples were randomly divided into three groups, and RNA was extracted within 3 days. The volume of plasma for RNA extraction was 2-3 ml by Norgen Plasma/Serum Circulating and Exosomal RNA Purification Kit (Slurry Format) (Cat. 42800) kit in a biosafety level 2 (BSL 2) laboratory at the department of inspection, east Hospital, people's Hospital of Wuhan University. 3 μL total RNA was used as input for miRNA library preparation by using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Catalog NO. E7300, New England Biolabs) following the manufacturer's instruction with modification, which labeled the pre-amplified small RNA molecules with unique molecular identifier (UMI)（LC-Bio Technology Co., LTD）. UMI reduces duplication bias in PCR and sequencing steps. The DNA library was purified by using 6 % PAGE gel, quantified by using QubitTM3 fluorometer (Invitrogen™, Cat. No. Q33216) with Qubit™ dsDNA HS Assay Kit (Invitrogen™, Cat. No. Q32854), examined by using Qsep100TM bio-fragment analyzer (Bioptic Inc., Taiwan, China), and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model. RNA libraries were prepared for sequencing using standard Illumina protocols