Maxima h minus

scRNA-seq libraries were prepared by amplifying the 3′ untranslated region (UTR) of the transcripts by using the modified CEL-Seq2 protocol (Hashimshony et al., 2016 (link)), which replaced the SuperScript II reverse transcriptase with Maxima H minus (EP0752; Thermo Fisher Scientific), the second strand synthesis reagent with the second strand synthesis module (E6111; New England BioLabs). A total of 384 cells in the same plate were pooled after reverse transcription. Each condition was analyzed in triplicate. Sequencing reads were obtained from HiSeq 1500 platform at the following cycles: 15 cycles of Read1 (Unique Molecular Identifier: 6 bp, cell barcode: 9 bp) and 36 cycles of Read2 (Illumina, San Diego, CA, United States), as shown in Supplementary Table S2.

mRNA expression levels in (co-)infected cells were assessed using real-time RT-qPCR. 500 ng of total RNA were reverse transcribed using an oligo(dT) primer and the enzyme Maxima H Minus (both from Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Next, qPCR was conducted using the Rotor-Gene Q real-time PCR cycler (Qiagen, Hilden, Germany) and the following primers: IFNB1, 5’-CATTACCTGAAGGCCAAGGA-3′ and 5′-CAGCATCTGCTGGTTGAAGA-3′; IFNL1, 5′-GGTGACTTTGGTGCTAGGCT-3′ and 5′-TGAGTGACTCTTCCAAGGCG-3′; MX1, 5′-GTATCACAGAGCTGTTCTCCTG-3′ and 5′-CTCCCACTCCCTGAAATCTG-3′; RSAD2, 5′-CCCCAACCAGCGTCAACTAT-3′ and 5′-TGATCTTCTCCATACCAGCTTCC-3′; GAPDH, 5′-CTGGCGTCTTCACCACCATGG-3′ and 5′-CATCACGCCACAGTTTCCCGG-3′ (all sequences from [37 (link)]); and DDX58, 5′-TGCAAGCTGTGTGCTTCTCT-3′ and 5′-TCCTGAAAAACTTCTGGGGCT-3′ [48 (link)]. The qPCR reaction mixture (10 μL) comprised 1× Rotor-Gene SYBR green PCR mix (Qiagen), 500 nM of each primer, and 4 μL of cDNA. DNA denaturation was conducted for 5 min at 95 °C, followed by 40 PCR cycles: 10 s at 95 °C and 20 s at 62 °C. Gene expression was calculated using the ΔΔCT method using GAPDH as the reference gene and expressed as fold change relative to untreated, uninfected cells.