The ST6GAL1 gene shRNA clone was obtained from
the MISSION shRNA library (Sigma Merck). Plasmid containing shRNA
or scrambled control was packaged into lentivirus using packaging
vectors pMD2.G and psPAX2 (packaging vectors were a kind gift from
Dr. Deepak K Saini). The plasmids were transfected into 293FT cells
(Thermo Fisher Scientific, R70007) using TurboFect (Thermo Fisher
Scientific, R0533). Cells were cultured in DMEM supplemented with
10% FBS; conditioned medium containing viral particles was collected
at 48 and 72 h. After filtering through a 0.45 μm filter, viral
particles were concentrated using the Lenti-X concentrator as mentioned
in the manufacturer’s protocol (TaKaRa, 631232). Concentrated
virus was aliquoted and stored at −80 °C until use. High
α2,6-Sial cells were seeded in a 24-well plate at 50–60%
confluence and transduced with viral particles containing shRNA or
scrambled control along with polybrene (4 μg/mL) for 24 h. After
72 h, transduced cells were selected using 5 μg/mL puromycin
(HiMedia, CMS8861). The knockdown of the gene was assayed using real-time
PCR and lectin flow cytometry as described earlier.
the MISSION shRNA library (Sigma Merck). Plasmid containing shRNA
or scrambled control was packaged into lentivirus using packaging
vectors pMD2.G and psPAX2 (packaging vectors were a kind gift from
Dr. Deepak K Saini). The plasmids were transfected into 293FT cells
(Thermo Fisher Scientific, R70007) using TurboFect (Thermo Fisher
Scientific, R0533). Cells were cultured in DMEM supplemented with
10% FBS; conditioned medium containing viral particles was collected
at 48 and 72 h. After filtering through a 0.45 μm filter, viral
particles were concentrated using the Lenti-X concentrator as mentioned
in the manufacturer’s protocol (TaKaRa, 631232). Concentrated
virus was aliquoted and stored at −80 °C until use. High
α2,6-Sial cells were seeded in a 24-well plate at 50–60%
confluence and transduced with viral particles containing shRNA or
scrambled control along with polybrene (4 μg/mL) for 24 h. After
72 h, transduced cells were selected using 5 μg/mL puromycin
(HiMedia, CMS8861). The knockdown of the gene was assayed using real-time
PCR and lectin flow cytometry as described earlier.