Monarch rna lysis buffer

A549 IRF1, 2, 3, 5, 7, 9, RIG-I KO clone, and A549 NT cells were seeded in a 24-well plate at a density of 1 × 10⁵ cells per well and stimulated with 500 IU/mL IFN-β (8499-IF-010/CF, R&D Systems) or 5 ng/mL IFN-λ1. The cells were washed once with PBS, directly lysed with 350 μL Monarch™ RNA Lysis Buffer (T2012L, NEB) per well at 8 and 24 h post stimulation and stored frozen at −80 °C until further analysis (see 2.6.3 Read-out by Quantitative RT-PCR).

Mouse fibroblasts were incubated with 500 U/mL mouse IFN-β protein (R&D Systems, Cat. # 8234 MB-010) in fibroblast media for 6 h at 37°C with 5% CO₂. After incubation, mouse IFN-β was removed and Monarch RNA Lysis Buffer (NEB, Cat. # T2012L) was added. RNA was isolated from fibroblast cell samples using the Monarch Total RNA Miniprep Kit (NEB, T2010S). RNA samples were analyzed in a one-step qRT-PCR using the Invitrogen EXPRESS Superscript One-Step qRT-PCR kit (Thermo Fisher Scientific, Cat. #11–781-01K). All reactions used either Taqman probe for Isg15 (Thermo Fisher Scientific, Mm01705338_s1), Ifit1 (Thermo Fisher Scientific, Mm00515153_m1), or Usp18 (Thermo Fisher Scientific, mM01188805_m1) and eukaryotic 18S rRNA endogenous control (Thermo Fisher Scientific, Cat. #4319413E), and were amplified on the Applied Biosystems QuantStudio 3 Real-Time PCR System.

Sorted cells were spun down and the pellet resuspended in Monarch RNA lysis buffer (New England BioLabs) supplemented with 2 mg/mL of Lysozyme (ThermoFischer). Bacteria grown in LB broth and harvested at the mid-logarithmic phase were used as control. Total RNA extraction was performed with the Monarch^® Total RNA Miniprep Kit (New England BioLabs) according to the manufacturers. RNA quality and quantity were assessed using a high-sensitivity RNA ScreenTape assay in a 4200 TapeStation (Agilent Technologies).
Libraries were prepared by using the SMARTer^® stranded total RNA-seq v2 pico input Mammalian (Takara) with minor technical adaptations. PCR1 was carried on for 5 cycles and PCR2 for 11 cycles (for the bacterial libraries) or 12 cycles (for the host–pathogen libraries). After cDNA synthesis, the human ribosomal cDNA was removed by using probes specific to mammalian rRNA.
Quality was assessed with a high-sensitivity DNA chip in a 4200 TapeStation. Finally, 750 ng of the resulting libraries were used for the enrichment protocol. Libraries were pooled in equimolar amounts. Sequencing was performed in the paired-end mode for 2 × 150 bp cycles using the Hiseq4000 and the NovaSeq6000 or the MiSeq sequencers (Oxford Wellcome Centre for Human Genetics) for dual RNA-seq or bacteria-only samples, respectively.