Dynamag 96 magnet

RNA was isolated from 60 µL of apical wash in 90 µL MagNA Pure External Lysis Buffer (cat. no. 06374913001, Roche). Lysates were stored at −20°C until further RNA extraction. In brief, lysates were incubated for 15 min with Agencourt AMPure XP beads (cat. no. 10453438, Beckman Coulter). After two washes with 70% ethanol on a DynaMag-96 magnet (Invitrogen), RNA was eluted in 50 µL Bidest water. Next, 5 µL of eluted RNA was used for the assessment of HMPV N expression by quantitative TaqMan real-time PCR in a similar approach as described before (32 (link)).

For determining the viral titer using qPCR, samples were thawed and centrifuged at 2000 g for 5 min. Sixty μL supernatant was lysed in 90 μL MagnaPure LC Lysis buffer (Roche) at RT for 10 min. RNA was extracted by incubating samples with 50 μL Agencourt AMPure XP beads (Beckman Coulter) for 15 min at RT, washing beads twice with 70% ethanol on a DynaMag-96 magnet (Invitrogen) and eluting in 30 μL MagnaPure LC elution buffer (Roche). Viral titers (TCID50 equivalents per mL) were determined by qRT-PCR using primer-probe sets described previously^{60 –62 (link)} and comparing the Ct values to a standard curve derived from a titrated virus stock.

For the measurement of gene expression by RT-qPCR, total RNA was isolated as described previously in Lamers et al. [46 (link)]. Briefly, 60 μL of sample was lysed in 90 μL of MagnaPure LC Lysis buffer (Roche) followed by a 30-min incubation with 50 μL Agencourt AMPure XP beads (Beckman Coulter). Beads were washed thrice with 70% ethanol on a DynaMag-96 magnet (Invitrogen) and eluted in 50 μL diethylpyrocarbonate treated water. In total 500 ng of RNA were reverse transcribed with SuperScript™ IV Reverse Transcriptase using Random Hexamer Primers according to the manufacturers protocol (Promega). Subsequently, gene expression was determined with SYBR GREEN PCR Mastermix (Applied Biosystems) according to the manufacturers protocol on a 7500 Real Time PCR Cycler (Applied Biosystems) with gene specific primers listed in Additional File 1: Table S2. Relative expression values were calculated with the 2^−ΔΔCT method and normalized to the average CT values of the housekeeping genes Gapdh and Rpl18. For quantification of SARS-CoV-2 specific RNA, a RT-qPCR targeting the E gene of SARS-CoV-2 was used as previously reported in Corman et al. [47 (link)] and Ct values were compared to a standard curve derived from a titrated D614G virus stock.

All samples were thawed and centrifuged at 2000x g for 5 min to spin down mucus and cellular debris. Supernatant was used for subsequent analysis. Virus titrations to determine pfu/ml were performed as described in S1 Methods for determining SARS-CoV-2 titres. RNA extraction was performed by adding 60 μl of sample to 90 μl MagnaPure LC Lysis buffer (Roche) for 10 minutes. Fifty μl Agencourt AMPure XP beads (Beckman Coulter) were added and incubated for followed by two washes on a DynaMag-96 magnet (Invitrogen) and elution in 30 μl ultrapure water. All steps were performed at room temperature. RNA copies were determined by qRT-PCR using primers targeting the E gene (51) and comparison to a standard curve.