Ifc chip

We used Fluidigm’s Biomark HD to validate lncRNA expression. Pre-amplified samples and primers were loaded onto a 96 by 96 integrated fluidic circuit (IFC) chip (Fluidigm, USA). Gene expression was quantified by the relative standard curve method^{31 (link)}, and using the SsoFast EvaGreen Supermix (Bio-Rad, 1725210, USA). LncRNA expression was normalized against the geometric mean of 3 HKG’s: ARHGEF12, B-ACTIN, and TUBA1A. Each of these 3 housekeeping genes was stable across conditions in both the RNA-seq and qPCR data (Supplementary Table 3). To assess their stability in qPCR data, each housekeeping gene was normalized to the geometric mean of the other 2 housekeeping genes.

Initially, the collected samples were screened for IAV by using the high-throughput rtPCR platform BioMark (Fluidigm, South San Francisco, USA) and the 192.24 Dynamic array (DA) integrated fluidic circuit (IFC) chip (Fluidigm). A 4 µL sample mix was prepared by mixing 2.2 µL of pre-sample mix (prepared by mixing 2 µL of 2X TaqMan Gene Expression Mastermix (Applied Biosystem) and 0.2 µL of 20X sample loading reagent (Fluidigm) for each sample) and 1.8 µL of pre-amplified sample. Similarly, 2 µL primer/probe stock was mixed with 2 µL of 2X assay loading reagent (Fluidigm). Three µL of the assay mix and 3 µL of sample mix was loaded into the respective inlets of the 192.24DA IFC chip. The 192.24 DA IFC chip was placed in the IFC controller RX for loading and mixing for approximately 30 min. Finally, the chip was inserted into the high-throughput rtPCR platform BioMark (Fluidigm) for thermal cycling with the following cycling condition: 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. All samples were tested in duplicates. Positive and non-template (nuclease-free water) controls were included. Amplification curves and cycle threshold (Ct) values were obtained on the BioMark system and finally analysed using Fluidigm Real Time PCR Analysis software 4.1.3 (Fluidigm) as previously described [41 (link)].