Cytoplasmic and nuclear RNA fractionation

Cytoplasmic and nuclear fractions were separated using PARIS^TM Kit (Cat no.: AM1921, Life, USA) according to the protocol. Briefly, about 1 × 10⁷ NPC cells were trypsinized and washed with ice-cold PBS. Next, 500 μL precooled cell fractionation buffer was added, and then cells were incubated on ice for 5 min. By centrifuging at 500×g for 5 min, we obtained the precipitation (nuclear fraction) and the supernatant (cytoplasmic fraction). The supernatant fraction was transferred into a new collection tube for further treatment, and the nuclear fraction was treated with 300 μL additional fractionation buffer to remove residual contamination. For obtaining nuclear and cytoplasmic fractions, 500 μL of lysis buffer was added and the tube was inverted five times. After that, the nucleus and cytoplasm lysate were separately mixed with an equal volume of 100% ethanol and pelleted with 14,000 rpm for one minute. After centrifuging and discarding the flow-through, the samples were rinsed in a 700-μL wash solution. The pure nucleus and cytoplasm RNA were eluted with a 40–60 μL elution solution at 100 °C. The expression of LINC-PINT was determined by RT-qPCR, and GAPDH and U6 were used as markers of cytoplasm and nucleus, respectively.