Nanodrop n1000

Total RNA was extracted from CD4⁺ and CD8⁺ lymphocytes using Trizol reagent (Gibco) and 1-Bromo-3-chloropropane (BCP) (Sigma Aldrich) in 2 ml heavy phase lock gel tubes (5 prime), according to manufacturer’s instructions. To reduce the risk of genomic DNA contamination, a DNase treatment was performed using DNase I (Invitrogen) and acid phenol chloroform (Sigma Aldrich). Sample quality control was performed using the Nanodrop N1000 (Fisher scientific) and Bioanalyzer 2100 (Agilent). RNA amplification of 400 ng total RNA was performed using the Illumina TotalPrep Amplification Kit (Ambion), according to manufacturer’s instructions. Biotin-labelled cRNAs were then hybridized to the HumanHT-12 v4 Expression (Illumina), according to manufacturer’s instructions.

Cells were lysed with RNA Lysis Buffer (PeqGOLD Total RNA kit, PeqLab), or TRIzol LS (Thermo Fisher Scientific), and kept at − 80 °C until total mRNA extraction, according to the suppliers instructions. Fetal liver (FL) pieces from wildtype slaughtered pig fetuses at day 25 of gestation, provided by FLI/Mariensee, were immediately flash frozen in liquid nitrogen and kept at − 80 °C until processing. FL pieces were first homogenized with TRIzol LS solution using stainless steel beads and processed by a TissueLyser LT device (Qiagen). The resultant homogenized solution was used for total mRNA extraction according to the TRIzol LS instructions protocol. Total mRNA from all samples was sub-sequentially treated with TURBO DNA-free Kit (Thermo Fisher Scientific), for complete removal of genomic DNA. The final product was quantified using the spectrophotometer Nanodrop N-1000 and processed for single stranded cDNA synthesis from 1 ug total mRNA in a final volume reaction of 10 uL using the High Capacity cDNA synthesis Kit (Thermo Fisher Scientific). The final cDNA was diluted 1:10 with RNAse and DNAse-free H₂O and used for qRT-PCR using the StepOnePlus device (AppliedBiosystems). All TaqMan probes used in this study are listed in Supplementary Table S3.

Bacterial DNA was extracted from the first vaginal swab using the PureLink Microbiome DNA purification kit (ThermoFisher Scientific, Carlsbad, CA, USA) according to the manufacturer´s instructions. Briefly, vaginal swabs were transferred from the -80 °C to the laboratory on dry ice and then placed in a 1.5 mL Eppendorf low-bind tube containing 800 µl of S1-Lysis Buffer. 100 µl of S2-Lysis Enhancer was added to each tube and vortexed, followed by a 10 min incubation at 65 °C and bead beat for 10 min on a vortex with a horizontal adapter. Swabs were not removed during the incubation and bead beating steps to maximize DNA extraction. Tubes were centrifuged for 1 min at 14,000 × g. Five hundred µl of the supernatant was transferred to a new tube and 900 µl of the S4-Binding buffer was added. After, the entire mix was loaded on to a spin column-tube assembly (2 × 700 µl) and centrifuged at 14,000 × g for 1 min. The spin column was washed by centrifugation with 500 µl of S5-Wash buffer and total DNA was eluted in 50 µl of S6-Elution buffer. DNA purity and quality were assessed by absorbance on a Nanodrop N1000 (ThermoFisher Scientific, Carlsbad, CA, USA) by measuring the A260/A280 ratio.

Fecal DNA was extracted at the System Biology Laboratory at the International Research Centre for HIV/AIDS Management and Prevention, Chantal Biya’s, Yaounde using PowerSoil DNA Isolation Kit according to the manufacturer instructions. Extracted DNA was transported frozen to the Lozupone lab at the University of Colorado, Anschutz Medical Campus Aurora Colorado for subsequent processing. Bacterial DNA concentration and purity was evaluated on a Nanodrop N1000 (Thermo Fisher Scientific, Carlsbad, CA, USA) by measuring the A260/A280 ratio. DNAs with an A260/A280 ratio of 1.8–2.0 were used for PCR amplification.

All the PCR products were purified for the NGS processing with the NucleoFast 96 PCR kit (Macherey-Nagel, Dueren, Germany). The purified product was quantitated by spectrophotometry using Nanodrop N1000 (Thermo Fisher, Wilmington, USA). Nucleic acids with concentrations adjusted to 0.2 ng/ul were sequenced on the Nextera XT (Illumina, CA, USA) sequencing platform.
The spike glycoprotein region located between 21709 and 23193 bps in the SARS-CoV-2 genome was aligned with the SARS-CoV-2 Wuhan Hu-1 isolate (GenBank accession number; MN908947.3). The primer pairs R: 5′-acacctgtgcctgttaaaacca-3′ and F: 5′-gacaaagttttcagatcctcagttttaca-3′. were used for sequencing [12 (link)], and sequencing was performed between the 118F–1652R primer region (∼1500 bp) on the Miseq sequencing platform (Illumina, CA, USA). Based on BWA software, all sequencing data were reassigned with Miseq Reporter (https://bio-bwa.sourceforge.net/). The protocol for NGS PCR sequencing was generated as follows: at 45°C for 10 min, at 95°C for 2 min, then for 40 cycles; 95°C for 10 s, 57°C for the 30 s, and 72°C for 30 s.

To characterize the gut microbiome, fecal samples were collected by study participants in a sterile container provided by the CIENI-INER shortly before their clinic visit and stored at −80 °C until DNA extraction. DNA was extracted using the QIAamp DNA stool minikit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Bacterial DNA concentration was measured by fluorometry using the Qubit^® dsDNA high-sensitivity assay kit and the Qubit^® 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA) as instructed by the manufacturer. DNA purity was also assessed by absorbance on a Nanodrop N1000 (Thermo Fisher Scientific, Carlsbad, CA, USA) by measuring the A260/A280 ratio. DNAs with an A260/A280 ratio of 1.8–2.0 were used for PCR amplification.