Author: Shifman, Ohad; Cohen-Gihon, Inbar; Beth-Din, Adi; Zvi, Anat; Laskar, Orly; Paran, Nir; Epstein, Eyal; Stein, Dana; Dorozko, Marina; Wolf, Dana; Yitzhaki, Shmuel; Shapira, Shmuel C.; Melamed, Sharon; Israeli, Ofir
Title: Identification and genetic characterization of a novel Orthobunyavirus species by a straightforward high-throughput sequencing-based approach Document date: 2019_3_4
ID: 15cxc32n_25
Snippet: RNA extraction and purification. RNA was purified from 140 µl of the cell culture supernatant by a QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's protocol, using 60 µl of AVE buffer for elution. As a control, RNA was extracted as mentioned above from noninfected cells. RNA was quantified using a Qubit fluorometer with the Qubit RNA HS Assay Kit (Invitrogen). The concentration of the purified RNA was 6 ng/µl (and 5 ng/µl fo.....
Document: RNA extraction and purification. RNA was purified from 140 µl of the cell culture supernatant by a QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's protocol, using 60 µl of AVE buffer for elution. As a control, RNA was extracted as mentioned above from noninfected cells. RNA was quantified using a Qubit fluorometer with the Qubit RNA HS Assay Kit (Invitrogen). The concentration of the purified RNA was 6 ng/µl (and 5 ng/µl for the control), which is sufficient for library preparation using the SMARTer Pico RNA Kit. BioAnalyzer (Agilent) analyses utilizing the RNA HS kit did not provide a relevant RNA integrity number (RIN) because the major RNA peak was generated from the carrier RNA, which is added as part of the QIAamp Viral RNA Mini purification procedure.
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