Dig rna labeling mixture

DIG-labeled cRNA probes used for Southern-blot or Northern-blot hybridization were transcribed from plasmid constructs including a full length SlartRDR6 sequence or a partial sequence of CaMV-35S promoter or a dimer of minus strand PSTVd-Dahlia (S1 and S2 Figs) [50 ]. The plasmid construct was digested with restriction enzymes and treated with phenol:chloroform (1:1, vol/vol). The ethanol precipitate was air-dried, suspended in ultrapure water, and used for in vitro transcription with T3 or T7 RNA polymerase and DIG RNA labeling mixture (Roche Diagnostics). The transcripts were collected by ethanol precipitation using LiCl, air-dried, and suspended in ultrapure water. Concentration and size of the probe was confirmed by agarose gel electrophoresis.

Primers LvTRIM9-1-pF1 with the T7 promoter and LvTRIM9-1-pR1 were designed to amplify a 551-bp fragment of LvTRIM9-1 as the template for sense probe synthesis. Primers LvTRIM9-1-pR2 with the T7 promoter and LvTRIM9-1-pF2 were designed for the template for antisense probe synthesis (Table S1). The PCR products were purified by MiniBEST DNA Fragment Purification Kit (Takara, Japan) and assessed by electrophoresis on 1.5% agarose gel. Digoxygenin (DIG)-labeled riboprobes were synthesized through in vitro transcription using DIG RNA Labeling Mixture (Roche, Mannheim, Germany) and TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific, USA). After assessing the concentration and quality of synthesized RNA probes by NanoDrop 2000 (Thermo Fisher Scientific, USA) and agarose electrophoresis, the DIG-labeled RNA probes were stored at -80°C for further use.
The paraffin-embedded lymphoid organ was sectioned into slices of 5–7 μm. Hybridization was performed following general protocol of the DIG RNA Labeling Kit (Roche, Germany). The concentration of both sense RNA probe and antisense RNA probe was 1 ng μl^-1. The signals were visualized by the color reaction using NBT/BCIP Stock Solution (Roche, Germany) and observed through a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).

The ascidian larvae were fixed in 4% paraformaldehyde for 2 h at RT. In situ hybridization was performed as previously described (Zhang et al., 2018 (link)). Labeled antisense RNA probes were transcribed from linearized DIG RNA Labeling Mixture (Roche America) according to the manufacturer’s instructions. The Lefty probe size is 780 bp, and the sequences of primers are as follows: forward primer, 5′ TTA​TCG​TCC​TGT​TCC​TCG​CA 3′ and reverse primer, 5′ GCT​GGT​TCC​TTC​ACG​TTT​GT 3′.

Eight probe primers (Table 2) were used to synthesize the RNA probe. The paired primers, P11 and P12, P15 and P16, were used to amplify the double-stranded DNA probe of chitin synthetase and chitinase, respectively. The T7 promoter sequence was added to the 5′ end of P13 and P17 primer, while the SP6 promoter sequence was added to 5′ end of P14 and P18. The double-stranded DNA probe with the T7/SP6 promoter sequence primer was amplified in order to add the promoter sequence for the in vitro transcription of single stranded RNA probe with T7/SP6 RNA polymerase and digoxin DIG RNA Labeling Mixture (Roche, Switzerland). The detailed step was conducted following the previously research (Thisse and Thisse, 2008 (link)).

Total RNA was isolated from Ca9-22 and HSC3 cells using NucleoSpin (Macherey-Nagel, Düren, Germany). Northern blot analysis was conducted using DIG Easy Hyb and an RNA probe made with Dig RNA labeling Mixture (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. RNA was reverse-transcribed to cDNA using oligo(dT) primers and M-MuLV reverse transcriptase (Thermo Fisher Scientific). PCRs were run with PrimeSTAR GXL DNA Polymerase (Takara) using the following thermal cycling program: 98°C for 1 min, 30 cycles of 98°C for 10 s, 58°C for 15 s, and 68°C for 20 s, and 68°C for 2 min. GAPDH was used for normalization. Quantitative RT-PCR was conducted using Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific) and a LightCycler Nano system (Roche Life Science) using the following thermal cycling program: 95°C for 10 min, 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. Primer sequences are available in S1 Table. For cDNA expression microarray analysis using SurePrint G3 Human GE 8x60K Ver.2.0 (Agilent Technologies, Santa Clara, CA, USA), total RNA was sent to a commercial service (Hokkaido System Science, Sapporo, Japan).