T7 high yield transcription kit

RNA probes for LoNA were in vitro transcribed using T7 High Yield Transcription Kit (Thermo Scientific), and then labeled with Alexa Fluor 488 or Alexa Fluor 594 on every G according to the manufacturer’s protocol (ULYSIS Nucleic Acid Labeling Kit, Invitrogen). RNA probes and cells were denatured at 80 °C for 10 min, and then incubated with probes for 24 h at 42 °C, followed by 2× SSC washing for 10 min at 45 °C. For immunostaining, slides after FISH were incubated with anti-NCL, anti-FBL, or anti-PolR1E antibodies for 3 h at 37 °C. Signals were detected and visualized using Alexa 594 or Alexa 488-labeled secondary antibodies (Invitrogen).

Viral nucleic acids from the blood samples were extracted using the RaPure Viral RNA/DNA Kit (Magen, China) according to the manufacturer’s instructions. Probe-based qPCR analysis was carried out using the AFD9600 Real-time System (AGS BioTech Co., China). Viral nucleic acids were eluted in 20 μl of nuclease-free water and stored at − 80 °C until use. For crRNA preparation, based on the conserved p72 sequence of ASFV, crRNAs were designed (Table S1) and cloned with a T7 promoter. The transcription templates were prepared by annealing the synthesized oligonucleotides. Then, crRNAs were transcribed and purified using a T7 High Yield Transcription Kit (Thermo Fisher Scientific) and an RNA Clean & Concentrator™-5 Kit (Zymo Research) respectively, each according to the manufacturer’s instructions. Finally, crRNAs were quantified using the Nanodrop 2000C (Thermo Fisher Scientific) and stored at − 80 °C.

The preparation of crRNA proceeded in three steps. The transcription templates for crRNA preparation were amplified by the PCR process, with the primers listed in Table 2. Then, the transcription process was performed at 37 °C overnight using the T7 High Yield Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the transcript products were purified using the RNA Clean & ConcentratorTM-5 (Zymo Research, Irvine, CA, USA) and quantified with NanoDrop 2000C (Thermo Fisher Scientific, Waltham, MA, USA).

Specific sequences for ABCB1 transporter transcripts were amplified from 8w human placental cDNA samples by PrimeSTAR GXL Polymerase PCR Supermix (Takara Bio, Mountain View, CA, USA) using primers complementary to the reference sequence NM_000927.4: ABCB1‐for 5′‐CAG TGT GCT GGA ATT CTT GGC AAA GCT GGA GAG ATC CTC‐3′ with ABCB1‐rev, 5′‐GAT ATC TGC AGA ATT CTG TAT CCA GAG CTG ACG TGG C‐3′. After purification, PCR fragments were recombined into an EcoRI‐linearized pCRII vector (Thermo Fisher Scientific, Mississaugua, ON, Canada) and transformed into competent Stellar™ E. coli cells using the In‐Fusion HD Cloning Kit (Takara Bio). Cloned fragments were verified for sense and antisense orientation by sequencing. For generation of digoxigenin (DIG)‐labelled RNA probes, vectors were linearized (ABCB1 antisense: EcoRV, sense: BamHI), phenol:chloroform purified and in vitro transcribed using the T7 High Yield Transcription Kit (Thermo Fisher Scientific) or the SP6 DIG RNA labelling kit (Roche, Canada). Labelled RNA probes were purified using the RNeasy Micro Kit (Qiagen, Canada), and labelling efficiency was analysed by dot blot analysis. In situ hybridizations were performed as described24 on 8 μm sections of 4% paraformaldehyde fixed, paraffin embedded placental tissues. Amounts of labelled probes were optimized for each probe preparation.

The protocol used for qPCR has been described in detail previously [28 (link),29 (link),40 (link)]. Briefly, 30 µL of cell culture (OD₆₀₀ = 0.2) were collected, treated with RNAprotect bacterial reagent (Qiagen, Germantown, MD, USA), centrifuged, and the resulting pellet was frozen at −80 °C. Once all samples were collected, RNA was extracted with RNeasy Mini kits (Qiagen, Germantown, MD, USA) according to instructions from the manufacturer. As an internal standard, 50 ng of purified phzM mRNA was added to each sample before extraction. phzM mRNA was generated by transcription with a T7 high yield transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) from a linearized pET11a plasmid containing phzM from P. aeruginosa PAO1. Extracted RNA was converted to cDNA with Taqman reverse transcription kits (Thermo Fisher Scientific, Waltham, MA, USA). Real-time qPCR was performed in a 0.1 mL MicroAMP fast optical 96-well reaction plate with SYBR green and appropriate primers (Table S2) in a ViiA 7 real-time PCR system for 40 cycles (Thermo Fisher Scientific, Waltham, MA, USA). The cycle threshold (Ct) value was used to calculate mRNA abundances in each sample. Calibration curves with pQE80-gfp_sf for gfp_sf and linearized pET11a-phzM for phzM were used to convert signals to DNA concentrations. gfp_sf cDNA was normalized by phzM for comparison between samples.

Total RNA extracted from A. coluzzii midgut infected with P. berghei c507 at 24 hpbf was used to prepare cDNA. The cDNA was used in conjunction with primers reported in (Habtewold et al., 2008 (link)) to amplify CTL4. DsRNA was then produced using the resulting PCR product and the T7 high yield transcription kit (ThermoFisher). 0.2 μg of purified dsRNA in 69 nl was injected into the thorax of A. coluzzii mosquitoes using glass capillary needles and the Nanoject II microinjector. Two to three days post injected mosquitoes were then infected with P. berghei.