Invitrogen superscript vilo cdna synthesis kit

In order to confirm the unreported variants detected by Genexus, we performed an independent sequencing analysis using Ion S5 XL System.
The cDNA was prepared using 7 µl of viral RNA by Invitrogen™ SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific, San Diego, CA).
Libraries were prepared using the Ion AmpliSeq SARS-CoV-2 Research Panel according to the Ion AmpliSeq™ Library Kit 2.0 user guide (Thermo Fisher Scientific, MAN0006735 rev F.0) and all the reactions were performed in an Applied Biosystems™ Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific, San Diego, CA). The final concentration of each cDNA library was determined on the Agilent 2100 system by the Agilent High Sensitivity DNA Assay (Agilent Technologies, Santa Clara, CA), following the manufacturer recommendations. Barcoded libraries were diluted to 30 pM, pooled in equal volume aliquots, and then loaded on to the Ion Chef™ Instrument (Thermo Fisher Scientific, San Diego, CA) for emulsion PCR, enrichment, and loading onto the Ion S5 520 chip. Two sequencing runs were performed on the Ion S5 XL System (Thermo Fisher Scientific, San Diego, CA). Reads were aligned with the Wuhan-Hu-1 NCBI Reference Genome (Accession number: MN908947.3) on the Torrent Suite v. 5.12.1.

Blood was collected for hormonal measurements and molecular analysis. Serum TSH was analyzed in a Cobas Roche Elecsys 600 instrument (Roche Diagnostics, IN, USA) with a detection sensitivity of 0.005 mIU/L and an interassay variance of 3.44%. The manufacturer’s reference range for TSH levels in pregnant women in the third trimester is 0.21 to 3.15 mIU/L. We also performed the assessment of serum TgAb (detection of 10–4000 mIU/L, reference range of less than 177.0 mIU/L), TPOAb (detection of 5–600 mIU/L, reference range of less than 123.0 mIU/L), and FT₄ (detection of 0.3–100 pmol/L, reference range of 0.65–1.21 ng/dL) in an Elecsys 2010 Roche Diagnostics instrument (Roche Diagnostics).
A PAXgene blood RNA tube (QIAGEN, NL, DE) was used for the preservation of RNA molecules. The extraction of total RNA was performed with the PAXgene Blood RNA Kit (QIAGEN, NL, DE), and we followed the manufacturer’s protocol. Total RNA was quantified with a Qubit fluorometer 2.0 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and the Invitrogen SuperScript VILO cDNA Synthesis Kit (Thermo Fischer Scientific) was used for cDNA synthesis.