Mirvana kit

Total RNA was isolated from neutrophils using miRVANA kit (Thermofischer Scientific, Waltham, MA, USA, #AM1560). cDNA was prepared from RNA using High-Capacity cDNA Kit (ABI, Thermo Fisher Scientific, Waltham, MA, USA, #4368813). mRNA expression of genes like DNAJC13, TMSB4X, AHSG, ATG5, ATG12, HMGB1, and HIF-1α were analyzed. The primer sequences of the genes are listed in Supplementary Table S5. 18s RNA served as endogenous control for normalization. Relative expression was analyzed using SYBR Green (Thermofischer Scientific, Waltham, MA, USA, #A25742) in a VIIA7 real-time PCR machine (ABI, Whitefield, Bangalore, India).

RNA was isolated from lung epithelial cells immediately after they were isolated, using the MirVana kit (AM1560, ThermoFisher). RNA quantity and quality was checked on a Nanodrop 1000 (ThermoFisher) and a BioAnalyzer (Agilent), respectively. RNA was amplified and biotinylated sense strand cDNA generated using the GeneChip WT Pico Reagent Kit (Affymetrix). cDNA was hybridised to GeneChip Mouse Transcriptome Assay 1.10 microarrays, using a GeneChip Fluidics Station (Affymetrix). Arrays were scanned using a GeneChip Scanner system (Affymetrix). Microarray data quality control was carried out using Qlucore software. Differential expression values were generated using the Affymetrix Transcriptome Analysis Console, and Ingenuity Pathway Analysis (Mar 2018 Release) was used for further data analysis. Following microarray analysis, differential expression of genes was confirmed using custom TaqMan Array Cards (ThermoFisher).

The fraction of small RNAs (<200 bp) of the C. gigas sample that was used to prepare the paired polyA and ribo-depleted libraries were extracted using the Mirvana kit (Thermofisher, Waltham, MA, USA). RNA was quantified by using a Qubit fluorimeter instrument, and the RNA size profile was determined with an Agilent small RNA chip (Agilent, Santa Clara, CA, USA). Library preparation and sequencing (PE150) was outsourced and carried out on an HiSeq Illumina platform (Admera Health, New York, NY, USA), and submitted to the NCBI SRA archive, under the accession ID SRR8587800. The paired reads were trimmed for quality, and for the presence of adaptors, as described for mRNA reads, and the correctly paired reads were joined into fragments. The resulting clean fragments, in a length range of 15–50 nt, were used for the detection of viral-derived small RNAs (vsRNAs) by direct mapping on the identified viral contigs or by using the VirusDetect pipeline [57 (link)]. To discriminate between genuine vsRNAs versus RNA degradation products, we correlated the number of mapped sRNA reads with the viral expression levels.

Cells were plated at 3 x 10⁵ cells per mL under normoxia or hypoxia and total RNA was extracted using mirVana kit (ThermoFisher Scientific). cDNA synthesis for mRNA analysis by q-PCR was performed using High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific) as per the manufacturer’s protocol on a T100 Thermal Cycler (Bio-Rad, Hercules, CA). The thermal conditions were 25°C for 10 minutes, 37°C for 2 hours, and 85°C for 5 minutes. qPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on a StepOnePlus Real-Time PCR System (Applied Biosystems). q-PCR reaction setup included enzyme activation at 95°C for 10 mins and 40 cycles of amplification at 95°C for 15 sec and 60°C for 1 min followed by melting curve analysis according to the instrument standard instructions. mRNA expression was normalized to that of 18S endogenous control RNA and the quantification of relative mRNA expression was performed using ΔΔCt method. The primers used for q-PCR were either synthesized by Lofstrand Labs Limited, Gaithersburg, MD or purchased from Bio-Rad (Hercules, CA) as ready-made assays. The sequences of primers synthesized and the assay numbers of the ones purchased from Bio-Rad are listed in S2 and S3 Tables, respectively.

Mouse ESCs (C57BL/6) were maintained on mitotically inactive mouse embryonic fibroblast feeders plus ESGRO LIF (Millipore); this cell line was recently authenticated and tested for contamination. Mouse ESC differentiation to NMPs was conducted as previously described (Gouti et al., 2014 (link); Turner et al., 2014 (link)) except for the use of retinoid-free B27 supplement that lacks both RA and retinoid substrates that could be used by the cells themselves to produce RA (retinyl esters, retinol, retinaldehyde, and carotenoids). The suppliers for critical reagents were as follows: N2 and retinoid-free B27 supplements (ThermoFisher), bFGF (PeproTech, Rocky Hill, NJ, USA), CHIR99021 (Cayman Chemical, Ann Arbor, MI, USA), all-trans RA (Sigma-Aldrich Corp.). Briefly, ESCs were grown at a density of 10⁴ cells cm⁻² as adherent cultures (using gelatin-coated 6-well Corning CellBind plates) in retinoid-free N2B27 medium with bFGF (10 ng/ml) for 48 h, then with N2B27/bFGF plus CHIR (5 µM) for 24 h (or N2B27/bFGF alone to test CHIR effect). At 72 h, bFGF/CHIR99021 was removed and replaced with N2B27 plus all-trans RA (25 nM, 1 µM, or DMSO control), then immediately processed for total RNA extraction (mirVana kit; ThermoFisher), which was purified and stored at −80°C.

Total RNA was isolated from 20 mg of tissue specimen by means of mirVana kit (ThermoFisher Scientific, USA—donors’ group) or Direct-zol RNA MiniPrep kit (Zymo Research, USA). The sample storage time varied from 1 to 42 months. RNA quality was verified by RIN (RNA integrity values) with 2100 Bioanalyzer (Agilent, USA) prior to further analysis. Reverse transcription was performed using SuperScript VILO Master Mix (Thermo Fisher Scientific, USA), using 500 ng of total RNA for 20 µL of reaction volume, according to a protocol from the supplier. Relative expression of transporter gene expression was determined by means of real-time PCR, using ViiA 7 Real-Time PCR System (Life Technologies, USA), TaqMan Fast Advanced Master Mix and pre-validated TaqMan assays (Thermo Fisher Scientific, USA). Threshold values were equal for all the genes, and C_T values were used to calculate relative transcript concentrations (ΔC_T method). Mean C_T value of five reference genes: GAPDH, PPIA, HMBS, RPLP0, and RPS9 was used as a reference for quantification of relative expression of the investigated DMEs genes. Details of TaqMan assays are provided in Supplementary Table 1.