In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly

Summary The synthesis of single-stranded riboprobes or double-stranded RNAs for in situ hybridization and gene knockdowns often use vectors that require time-consuming plasmid restriction digests and inefficient gel purifications. Here, we present a faster protocol for the simultaneous plasmid restriction digestion and Gibson assembly of vectors for the synthesis of both riboprobes and double-stranded RNAs for in situ and RNA interference experiments, respectively. We illustrate the protocol with planaria in situ and RNAi assays, but it is applicable to any organism.

vi. 300 mL 100 mM b-NAD. b. Vortex to mix. c. Add sterile water to 6 mL with p1000 pipettor (about 1 mL). d. Vortex to mix. e. Store in 18 aliquots of 320 mL each in 1.5 mL tubes at À20 C. The aliquots remain stable for at least 1 year at À20 C. 5. Prepare 23 Gibson Master Mix. a. Keep all tubes on ice or in À15 C mini-cooler at all times. b. Add to 1.5 mL tube with 320 mL of 53 Gibson Reaction Buffer: i. 0.64 mL T5 Exonuclease.
iii. 160 mL Taq DNA Ligase. iv. 300 mL sterile water. c. Pipet up and down to mix. d. Store in 80 aliquots of 10 mL each in 0.2 mL PCR tubes at À20 C (keep them on ice during aliquoting). The aliquots remain stable for at least 1 year at À20 C. Planarian total RNA will be extracted from worms pooled from different regenerative time points as well as whole worms. Be sure to cool down the centrifuge to 4 C in advance.

KEY RESOURCES
1. Place worms in a 2 mL tube. Remove as much residual water as possible.
CRITICAL: If using another organism, be sure to include stages/time points where the gene of interest is expressed to ensure that its transcript will be included in the total RNA pool after extraction. We pool together multiple stages/time points to increase this likelihood.
2. Add 500 mL TRIzol reagent per 2-3 worms and place at À80 C for at least 1 h to quick-freeze them.
CRITICAL: TRIzol reagent is a toxic chemical and should be handled and disposed of safely.
3. Thaw the lysate at 20 C-22 C for 30 min to 1 h, vortexing every 10 min. 4. Centrifuge the lysate at 12,000 3 g for 5 min at 4 C. Transfer the clear supernatant to a new tube. 5. Incubate for 5 min at 20 C-22 C to permit complete dissociation of the nucleoproteins complex. 6. Add 200 mL chloroform for 1 mL of TRIzol reagent used for lysis, then securely cap the tube. Vortex for 1 min.
CRITICAL: Chloroform is a toxic chemical and should be handled and disposed of safely.
7. Incubate for 3 min at 20 C-22 C. Centrifuge the sample at 12,000 3 g for 15 min at 4 C. 8. Transfer the upper aqueous phase containing the RNA to a new 2 mL tube by angling the tube at 45 and pipetting the solution out. Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase. 9. Add 500 mL isopropanol per 1 mL of TRIzol reagent used for lysis to the aqueous phase. Mix for 2 s and incubate for 10 min at 20 C-22 C. 10. Centrifuge for 10 min at 12,000 3 g at 4 C. Total RNA precipitate forms a white gel-like pellet at the bottom of the tube. Discard the supernatant without disturbing the pellet.
Luria Broth (LB): Mix with 500 mL of deionized water and transfer to a 1 L bottle and autoclave.

Reagent Amount
Tryptone 5 g Yeast extract 2.5 g NaCl 5 g LB Agar: Mix with 100 mL of deionized water and transfer to a 250 mL bottle and autoclave.

Reagent Amount
Tryptone 1 g Yeast extract 0.5 g NaCl 1 g Agar 1.5 g ll OPEN ACCESS 11. Add 1 mL of 75% ethanol for 1 mL of TRIzol reagent used for lysis. 12. Vortex the sample for 2 s, then centrifuge for 5 min at 7,500 3 g at 4 C. Discard the supernatant without disturbing the pellet. 13. Air dry the RNA pellet for 5-10 min. Do not dry for too long, as the pellet will be difficult to resuspend. 14. Resuspend the pellet in 20-50 mL nuclease-free water by pipetting up and down. The resuspension will be dark in color. 15. Incubate in a heat block set at 55 C-60 C for 10-15 min. 16. Quantify the RNA concentration using a NanoDrop spectrophotometer or Agilent's Bioanalyzer. 17. Store the RNA at À80 C or proceed with cDNA synthesis.
Note: This protocol is adapted from (Liu and Rink, 2018) and  as well as the manufacturer's instructions for the TRIzol reagent.

cDNA synthesis
Timing: 1 h RNA is converted to cDNA to use as a template to synthesize the insert for the gene of interest in the Gibson assembly.
18. Thaw all components except the Reverse Transcriptase thoroughly and centrifuge for 2 s to collect components at the bottom of the tube before using. Place components on ice. 19. Add the following components to a 0.2 mL PCR tube in the indicated order: 20. Add the following components in the indicated order: 21. Mix gently and centrifuge for 2 s. Incubate the tube as follows in a thermal cycler: 22. The reaction product can be used directly in PCR applications or stored at À20 C for less than one week. For longer storage, À80 C is recommended. Step 1 5 min at 25 C Step 2 60 min at 42 C Step 3  Note: This protocol is adapted the manufacturer's instructions for the RevertAid Reverse Transcriptase kit. Alternatively, any other cDNA synthesis kit will work here.

cDNA amplification
Timing: 4 h Using gene-specific primers with Gibson overhangs, the insert for the gene of interest is cloned from cDNA.
23. Prepare the PCR master mix on ice: 24. Vortex the tubes for 2 s to mix. Run the tubes in a thermal cycler with the following conditions: Note: Annealing temperature will depend on the primers you have designed. We recommend using the calculator from the polymerase vendor or your thermal cycler's gradient feature to find the suitable annealing temperature for your primer pair.
25. Run 1-3 mL of the PCR(s) on an agarose to confirm a successful PCR. 26. Clean the PCR(s) according to the manufacturer's instructions for the PureLink PCR Purification Kit. a. Add 4 volumes of PureLink Binding Buffer HC (B3) to 1 volume of the PCR product. Mix well. b. Add the sample to a PureLink Spin Column. c. Centrifuge the column at 20 C-22 C at 10,000 3 g for 1 min. d. Discard the flow through and place the spin column into the collection tube. e. Add 650 mL of Wash Buffer to the column. f. Centrifuge the column at 20 C-22 C at 10,000 3 g for 1 min. Discard the flow through from the collection tube and place the column into the tube. g. Centrifuge the column at maximum speed at 20 C-22 C for 2-3 min to remove any residual Wash Buffer. Discard the collection tube. h. Place the spin column in a clean 1.7 mL PureLink Elution Tube provided with the kit. i. Add 50 mL of Elution Buffer (or sterile water) to the center of the column. j. Incubate the column at 20 C-22 C for 1 min. k. Centrifuge the column at maximum speed for 2 min. l. Remove and discard the column (the recovered elution volume is $48 mL).
Note: The PureLink purification kit has a buffer (B3) that allows for the removal of PCR products smaller than 300 bp. When running the PCR products on a gel, you will likely observe small bands distinct from the correctly-sized bands produced by the appropriate PCR amplification.
We have found that small products (likely small primer products) interfere with subsequent Gibson reactions, so it is important to use Buffer B3 when purifying your PCR products.
27. Quantify the concentration of the PCR product using your preferred method (i.e., Nanodrop, spectrophotometer, etc.). 28. Store the gene insert PCR at À20 C until ready to proceed with Gibson assembly.
Alternatives: High-fidelity polymerases (e.g., Phusion, Q5) are recommended to ensure high accuracy in the synthesis of your gene insert for the subsequent downstream nucleic acid synthesis products (e.g., riboprobes) to be as close to the target mRNA sequence as possible. However, we have also synthesized successful in situ riboprobes using Taq-based polymerases.

Gibson assembly and bacterial transformation
Timing: 2 days The PCR product from the gene of interest will be inserted into the pDL1 vector. The restriction digest of the vector and the Gibson reaction to insert the PCR product is done in the same tube.
Note: The pDL1 plasmid (Figure 1) can be purchased through Addgene (cat #182263). Similar to plasmid pJC53.2 for T/A Cloning (Collins et al., 2010), pDL1 includes T3 and SP6 promoters for single-stranded RNA synthesis, and two T7 promoters and terminators for dsRNA synthesis. In addition, pDL1 includes a MCS region flanked by BspQI asymmetrical restriction sites, M13 forward and reverse universal sequences before the T7 terminators for complete forward and reverse sequencing of the inserted gene, and the AmpR resistance gene. pDL1 was constructed by Gibson assembly of PCR-amplified pGEM-5Zf(+) (Promega) backbone with two synthesized gBlocks (IDT), one for each T7 terminator-M13-promoters-restriction sequence due to the presence of the two inverted T7 promoters. In the design of the gBlock sequences, one base was added between the M13 promoters and T7 terminators to prevent hairpins in the M13 promoters and reduce annealing between the two opposite T7 terminator/promoter pairs. Finally, the T3 and SP6 promoter sequences are compatible with their universal primers, as an alternative to the M13 primers for sequencing.
29. Calculate the volume needed of insert for a 3:1 molar ratio with 10-100 ng of pDL1 backbone (2196 bp after digestion of pDL1 with BspQI) as: insert ml = 3, backbone ml , backbone ng=ml insert ng=ml , insert bp backbone bp We regularly use 2 mL of vector diluted at 12.5 ng/mL, resulting in: a. Calculated mL pDL1 backbone. b. Calculated mL insert DNA (experimental) OR equivalent mL nuclease-free water (control). c. Nuclease-free water up to 10 mL total. d. 0.5 mL BspQI.
Note: BspQI cuts at the same site as SapI but at 50 C (the same temperature as the Gibson reaction), allowing the digest and the Gibson assembly to occur simultaneously in the same tube. Crucially, the restriction digest produces an asymmetrical cut, preventing the vector from re-linking. Thus, this results in very low background.

Plasmid purification and verification
Timing: 2 days The completed plasmid will be grown and purified and then sequenced to verify that the insert from the gene of interest has been inserted into the backbone.
38. Prepare 15 mL LB-Ampicillin liquid media and divide equally into three 50 mL Falcon tubes. LB media can be stored at 20 C-22 C for 3-4 months. 39. Add a different single colony from the experimental LB-Ampicillin plate to each tube. 40. Incubate the tubes in a shaker at 37 C at 400 rpm for 12-18 h.
Note: The shaking rotational speed indicated is for a mini-shaker. It should be adjusted according to your particular shaker's orbital diameter for standard bacterial culture growth. ll

OPEN ACCESS
f. Add 250 mL of Lysis Solution and invert the tube 4-6 times until the solution becomes viscous and slightly clear. g. Add 350 mL of Neutralization Solution and invert the tube 4-6 times. The neutralized bacterial lysate should become cloudy. h. Centrifuge at 12,000 3 g for 5 min to pellet cell debris and chromosomal DNA. i. Transfer the supernatant to the supplied GeneJET spin column by pipetting. Avoid disturbing or transferring the white precipitate. j. Centrifuge at 12,000 3 g for 1 min. Discard the flow through and place the column back into the same collection tube. k. Add 500 mL of Wash Solution to the GeneJET spin column. Centrifuge at 12,000 3 g for 30-60 s and discard the flow through. Place the column back into the same collection tube. l. Repeat the wash procedure (previous step) using 500 mL of Wash Solution. m. Discard the flow through and centrifuge for an additional 1 min at 12,000 3 g to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. n. Transfer the GeneJET spin column into a fresh 1.5 mL tube. Add 50 mL of Elution Buffer to the center of the spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at 20 C-22 C and centrifuge at 12,000 3 g for 2 min. o. Discard the column.
Alternatives: Any plasmid miniprep kit can be used here to purify the plasmids.
42. Quantify the plasmid concentrations. Purified plasmids can be stored at À20 C. 43. Run 1-3 mL of the 3 plasmids on an agarose gel along with the original pDL1 vector to observe which plasmid had a successful Gibson reaction. A successful Gibson will result in the brightest band not running as far as the brightest band of pDL1. Note: More colonies can be grown if none of the 3 initial colonies produced a plasmid with a successful Gibson reaction. However, the Gibson reaction and digest are very efficient, so this is very unlikely.

Synthesis of RNA probes for in situ hybridization
Timing: 16 h The created vector is used to synthesize an RNA probe for the purpose of in situ hybridization. Alternatives: Any PCR purification kit can be used here.
e. Quantify the concentration of the PCR product using your preferred method (i.e., Nanodrop, spectrophotometer, etc.). Store the remainder at À20 C until ready for riboprobe transcription.
Alternatives: As with above, high-fidelity polymerases (e.g., Phusion, Q5) are recommended to ensure high accuracy in the amplification of the PCR product that serves as the template for riboprobe synthesis.
46. Prepare the following transcription reaction mixture at 20 C-22 C in 0.2 mL PCR tubes: CRITICAL: When synthesizing the riboprobe, work in an RNase-free environment and use RNase-free reagents/tubes whenever possible to prevent RNA degradation.
Note: The transcription buffer is included with the enzyme (T3 or SP6). Ensure the transcription buffer is thawed and fully dissolved.
Note: Using SP6 polymerase will result in the synthesis of a probe anti-sense to the gene insert sequence, while T3 polymerase will yield a probe sense to the gene insert. Both sense and anti-sense riboprobes can then be used in subsequent in situ hybridization experiments, with anti-sense probes used to detect the expression of the gene of interest and sense probes used as negative control.
47. Vortex the tubes for 2 s to mix and centrifuge the reaction(s 62. Carefully remove the supernatant with a pipet without disturbing the pellet. 63. Air-dry the pellet for a few minutes. Do not over-dry the RNA pellet as this will make it difficult to resuspend. 64. Resuspend the pellet in 100 mL de-ionized formamide.
CRITICAL: Formamide is a toxic chemical and should be handled and disposed of safely.
65. Analyze 2 mL of the resulting riboprobe(s) by electrophoresis on a gel (as RNase-free as possible). 66. Store the remainder of the riboprobe(s) at À80 C.
Alternatives: Here we have outlined the synthesis of a digoxigenin (DIG)-labeled riboprobe. Other labels can be used to create riboprobes, such as fluorescein and dinitrophenol (DNP).

Timing: 16 h
The created vector is used to synthesize a double-stranded inhibitory RNA molecule for the purpose of gene knockdown.
67. Generate the DNA template for dsRNA transcription.
a. Prepare the following PCR on ice: b. Vortex the tubes for 2 s. Run the tubes in a thermal cycler with the following conditions: PCR cycling conditions c. Run 1-3 mL of the PCR products on an agarose gel to confirm a successful PCR. d. Purify the PCR products with the Zymo Research DNA Clean & Concentrator-5 kit (as outlined above).
Alternatives: Any PCR purification kit can be used here. e. Quantify the concentration of the PCR product using your preferred method (i.e., Nanodrop, spectrophotometer, etc.). f. Store the remainder at À20 C until ready for riboprobe transcription. 68. Prepare the following transcription reaction mixture at 20 C-22 C in 0.2 mL PCR tubes: Note: The transcription buffer is included with the enzyme (T7). Ensure the transcription buffer is thawed and fully dissolved.
69. Vortex the tubes for 2 s to mix and centrifuge the reaction(s). 70. Incubate the reaction(s) in a thermal cycler/water bath at 37 C for 12-18 h. 71. Add 5 mL DNase I. Incubate at 20 C-22 C for 20 min. 72. Transfer the reaction(s) to new (RNase-free) 1.5 mL tubes. 73. Add 55 mL 5 M ammonium acetate. 74. Add 275 mL ice-cold 100% ethanol. Mix well. 75. Incubate at À20 C for 1 h. 76. Centrifuge at max speed for 15 min at 4 C. 77. Carefully remove the supernatant with a pipet without disturbing the pellet. 78. Wash the pellet with 700 mL ice-cold 70% ethanol. 79. Centrifuge at max speed for 15 min at 4 C. 80. Carefully remove the supernatant with a pipet without disturbing the pellet. 81. Air-dry the pellet for 5 min at 20 C-22 C. Do not over-dry the RNA pellet as this will make it difficult to resuspend. 82. Resuspend the pellet in 75 mL nuclease-free water. 83. Analyze 2 mL of the resulting dsRNA by electrophoresis on a gel. A lower percent gel such as 0.8% works well here. 84. Quantify the concentration of the dsRNA using your preferred method (i.e., Nanodrop, spectrophotometer, etc.). 85. Store the remainder at À80 C.

EXPECTED OUTCOMES
This protocol is designed to generate a plasmid vector suitable for the synthesis of antisense riboprobes for in situ hybridization and dsRNA for RNA interference (RNAi). Using vectors synthesized by our lab, we have successfully made riboprobes to examine in Schmidtea mediterranea the expression of genes using colorimetric in situ hybridization and synthesized dsRNA for RNAi against several genes to inhibit their expression. The use of plasmids generated using this protocol can be applied to other organisms. In general, we recommend that you utilize conventional in situ and RNAi methodology for your particular organism, including riboprobe length and stages for pooling RNA for cDNA synthesis. Additionally, concentrations of templates for riboprobe dsRNA synthesis can be altered to synthesize these RNA products of a concentration appropriate for your particular system.  Figure 2 outlines the features of a successfully synthesized plasmid and its applications using the presented protocol. The gene-specific sequence is flanked by the promoters for T3 (sense) and SP6 (antisense) RNA polymerases. These promoters are used to synthesize sense and antisense riboprobes, respectively, containing a sequence specific for a particular gene for in situ hybridization. Flanking those promoters are promoter and terminator sequences for T7 polymerase, which can be used for the synthesis of double-stranded inhibitory RNA containing the gene-specific sequence. To illustrate the protocol, we synthesized a plasmid for planaria Smed-Slit-1 (pAW2-Smed-Slit-1; Addgene, 182264) and used it to generate a sense and antisense riboprobe, as well as a dsRNA to inhibit Slit-1 expression. Performing in situ hybridization using the synthesized riboprobes, we detected a midline expression ( Figure 2C) with the antisense probe and no expression ( Figure 2B) with the sense probe in whole worms. Upon delivery of dsRNA for Slit-1 and amputation of the head, we observed the collapse of the medio-lateral axis, signified by the regeneration of a single eye at the midline of the worm ( Figure 2E, arrow) compared to worms fed control dsRNA that regenerate two eyes (Figure 2D, arrows. Both the expression pattern and knockdown phenotype of Slit-1 obtained are similar to previously published (Cebrià et al., 2007), validating the presented streamlined protocol.

LIMITATIONS
A potential limitation of the outlined protocol is knowing the sequence of the gene of interest. If the sequence is not known or is poorly annotated, primers cannot be designed.

TROUBLESHOOTING
Problem 1 Insufficient RNA is isolated.

Potential solution
The listed procedure is designed for isolating RNA from planarian worms. Reaction component volumes, concentrations, and incubation times may need to be adjusted for isolating RNA from other organisms to produce enough RNA for the subsequent steps.

Problem 2
Primers with overhangs do not amplify cDNA template for PCR.

Potential solution
The long overhangs can make PCR amplification difficult for some genes, as they are much more prone to creating self-dimers and hetero-dimers which can impede PCR. When designing the primers, ensure that they have low Delta G values in regard to self-dimers and hetero-dimers for the pair (ideal Delta G is > 7 kcal/mole). Changing the cycling conditions based on recommendations by the manufacturer of the enzyme may help as well (e.g., altering the extension time).

Problem 3
Gibson reaction not successful.

Potential solution
Ensure that any small primer products are removed when cleaning the PCRs. In our hands, these small products can hinder the Gibson reaction and prevent the correct insertion of the gene insert. We highly recommend using the B3 Buffer in the PureLink Purification Kit that is outlined in the protocol. To be certain the small primer products are removed, run the purified PCR on a gel as well.

Problem 4
Sense riboprobe negative control results in high background signal.

Potential solution
Decreasing background signal will depend on the animal that you are using, as preparation of the tissue varies from species to species. Reducing the amount of riboprobe added could reduce the background. Another cause could be that the sense probe is binding to transcripts from the opposite strand, though it is highly unlikely. BLAST the sequence of the sense probe against the transcriptome to see if any matches exist. Alternatively, probes from an unrelated species can be used as negative control.

Problem 5
RNAi with dsRNA results in no phenotype.

Potential solution
Increasing the dosage of dsRNA, either by concentration or number of deliveries of RNA, could produce the desired phenotype. Alternatively, you can use a different delivery method of dsRNA (e.g., injection vs. feeding).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Daniel Lobo (lobo@umbc.edu).

Materials availability
Plasmids generated in this study have been deposited to Addgene.

Data and code availability
This study did not generate or analyze any datasets or code.