Wash solution a

A portion of the sample was used for transcriptomic analysis. The CTC-enriched sample was centrifuged at 300× g for 10 min to pellet the cells. The cell pellet was lysed in 700 µL of TRIzol^TM Reagent (Life Technologies, Carlsbad, CA, USA) and incubated for 5 min at room temperature. The lysed sample was immediately frozen at −20 °C for up to 2 weeks before RNA isolation and purification.
RNA was purified using an in-house, modified protocol using the Total RNA Purification kit (Norgen Biotek Corp., Thorolod, ON, CA). The protocol utilized TRIzol^TM Reagent to lyse the sample to increase RNA yield in the presence of the remaining blood components, mainly RBCs. After the sample in TRIzol^TM Reagent was thawed, 140 µL of chloroform was added and vortexed to mix. After 3 min of incubation at room temperature, the sample was centrifuged at 12,000× g for 15 min. The aqueous layer (RNA layer) was collected and mixed with equal volume 70% ethanol and added to the provided columns (Norgen Biotek Corp., Thorolod, ON, CA). The columns were washed 3× with the provided Wash Solution A (Norgen Biotek Corp., Thorolod, ON, CA), and eluted into 30 µL of provided Elution Solution A (Norgen Biotek Corp., Thorolod, ON, CA).
All purified RNA, cDNA, and PCR reagents were handled in a PCR workstation to prevent nuclease contamination.

To determine the subcellular localization of RNA, fractionation of nuclear and cytoplasmic RNA was performed by using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada). Cells were first lysed with Lysis Buffer J (Norgen Biotek), and the lysate was separated through centrifugation, with the supernatant containing the cytoplasmic RNA and the pellet containing the nuclear RNA. Buffer SK (Norgen Biotek) and ethanol were then added to the cytoplasmic and nuclear fractions, and the solution was loaded onto a spin column to collect RNA. The bound RNA was then washed with Wash Solution A (Norgen Biotek) to remove the remaining impurities, and the purified RNA was eluted with Elution Buffer E (Norgen Biotek). The isolated RNA was subsequently reverse-transcribed, and the relative expression level was measured by qPCR. The pairs of primers used are listed in Table S3.

To determine the subcellular localization of RNA, fractionation of nuclear and cytoplasmic RNA was performed using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada). Cells were first lysed with Lysis Buffer J (Norgen Biotek), and the lysate was separated by centrifugation, after which the supernatant contained the cytoplasmic RNA and the pellet contained the nuclear RNA. Buffer SK (Norgen Biotek) and ethanol were then added to the cytoplasmic and nuclear fractions, and the solution was loaded onto a spin-column to collect RNA. The bound RNA was then washed with Wash Solution A (Norgen Biotek), and the purified RNA was eluted with Elution Buffer E (Norgen Biotek). The isolated RNA was subsequently reverse-transcribed, and the relative expression level was measured by qPCR. The pairs of primers used are listed in Table 1.