Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400×g) in a table-top microcentrifuge. Discard the flowthrough, and set the Spin Column CP3 back into the collection tube.

2. Harvest 5 ml bacterial cells in a microcentrifuge tube by centrifugation at 4,000 rpm for 10 min at room temperature (15-30°C), then remove all traces of supernatant by gently inverting the open centrifuge tube until all medium has been drained.

3. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to 1.5 mL microcentrifuge tubes.

4. Add 250 μl Buffer P2 and invert the tube 6-8 times.

5. Add 350 μl Buffer P3 and invert the tube 6-8 times.

6. Centrifuge for 10 min at 12,000 rpm (~13,400×g)

7. Carefully transfer the supernatant from step 6 to the Spin Column CP3 by decanting or pipetting. Centrifuge for 1 min at 12,000 rpm (~13,400×g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube

8. Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure the ethanol (96%-100%) has been added to Buffer PW) and centrifuging for 30-60 sec at 12,000 rpm (~13,400×g). Discard the flow-through and set the CP3 back into the Collection Tube.

9. Wash Spin Column CP3 by adding 600 µl Buffer PW and centrifuging for 1 min at 12,000 rpm (~13,400×g).

10. Discard the flow-through, and centrifuge for an additional 2 min at 12,000 rpm (~13,400×g) to remove residual wash Buffer PW.

11. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 μl Buffer TB or water (pH7.0-8.5) to the center of the Spin Column CP3, let it stand for 2 min, and then centrifuge for 2 min at 12,000 rpm (~13,400×g)

2.1.2 PCR amplification

1. Label 7 microcentrifuge tubes (See table)

2. Add 25 μL mix, 2 μL forward primer, 2 μL reverse primer, 2 μL DNA template, and 19 μL water to each tube.

Fragment Name	DNA Template	Primers (F/R)	Length/bp	Annealing Temp/°C
Type9k-F1	P-Type9k	Type9k-gib-F1	3353	56
		Type9k-gib-R1
Type9k-F2	P-Type9k	Type9k-gib-F2	2872	56
		Type9k-gib-R2
F-Fel d1-CH1	P-Fel d1-CH1	d1CH1-gib-F	481	56

		d1CH1-gib-R
F-Fel d1-CH2	P-Fel d1-CH2	d1CH2-gib-F	534	56
		d1CH2-gib-R
F-Fel d2	P-Fel d2	d2-Gib-F	1944	56
		d2-Gib-R
F-Fel d4	P-Fel d4	d4-Gib-F	918	56
		d4-Gib-R
F-Fel d7	P-Fel d7	d7-Gib-F	798	56
		d7-Gib-R

Run PCR with the following settings:

Step

Temperature (°C)

Time

Cycles

Initial Denaturation

95

5 min

1 cycle

Denaturation

95

30 s

Repeat steps 2-4 for 40 cycles

Annealing

56

30 s

Extension

72

X min

(see table for details)

Final Extension

72

10 min

1 cycle

Hold

4

Indefinite

1 cycle

2.1.3 Electrophoresis

Materials	Quantity
1xTAE Buffer	40 mL
Agarose	0.6 g
SYBR Gold	4 μL
6X Loading Buffer	8 μL per PCR product tube
DNA Markers	DL 2000

Mix 40 mL 1xTAE Buffer with 0.6 g agarose in a conical flask.
Heat in a microwave until dissolved.
Cool and add 4 μL SYBR Gold mix well.
Pour into a gel casting tray, let solidify.
Add 6X loading buffer to PCR products
Load samples into gel wells.
Load 10 μL DL2000 marker
Run electrophoresis at 180 V for 15 min.
Visualize the gel under UV light.

2.1.4 Gel extraction

We use Universal DNA Purification Kit (GDP214) for gel extraction.

1.Column equilibration: Add 500 μl Buffer BL to the Spin Column CB2 (in a 2 ml Collection Tube). Centrifuge at 12,000 rpm (~13,400 × g) for 1 min.

2. Cut the target DNA band from the agarose gel and transfer it into a clean centrifuge tube, and then weigh the gel block.

3. Add equal volume of Buffer PC to the volume of gel.

4. Incubate at 50°C for about 10 min by inverting up and down the tube until the agarose gel dissolved completely.

5. Transfer the whole solution to the Spin Column CB2. Centrifuge at 12,000 rpm (~13,400 × g) for 1 min.

5. Add 600 µl Buffer PW (Ensure that ethanol (96-100%) has been added to Buffer PW before use) into the Spin Column CB2 and centrifuge at 12,000 rpm (~13,400 × g) for 1 min.

6. Repeat Step 5.

7. Set the Spin Column CB2 back to the Collection Tube and centrifuge the column at 12,000 rpm (~13,400 × g) for an additional 2 min to remove residual wash buffer. Discard the flow-through, and allow the column to air dry with the cap open for 2-5 mins to dry the membrane.

8. To elute DNA, place the column in a clean 1.5 ml microcentrifuge tube.

Add appropriate volume of Buffer EB (For fragments >4 kb, Buffer EB should be preheated to 65-70°C) to the center of the membrane, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm (~13,400 × g) for 2 min.

2.1.5 Gibson Assembly

Set up the following reaction on ice:

Type 9k-Fel d1-CH1
Materials	Usage
Gibson Assembly Mix	5 ul
Type9k-F1	70 ng
Type9k-F2	65 ng
F-Fel d1-CH1	80 ng
dd H₂O	Up to 10 ul

Type 9k-Fel d1-CH2
Materials	Usage
Gibson Assembly Mix	5 ul
Type9k-F1	70 ng
Type9k-F2	65 ng
F-Fel d1-CH2	80 ng
dd H₂O	Up to 10 ul

Type 9k-Fel d2
Materials	Usage
Gibson Assembly Mix	5 ul
Type9k-F1	70 ng
Type9k-F2	65 ng
F-Fel d2	125 ng
dd H₂O	Up to 10 ul

Type 9k-Fel d4
Materials	Usage
Gibson Assembly Mix	5 ul
Type9k-F1	70 ng
Type9k-F2	65 ng
F-Fel d4	60 ng
dd H₂O	Up to 10 ul

Type 9k-Fel d7
Materials	Usage
Gibson Assembly Mix	5 ul
Type9k-F1	70 ng
Type9k-F2	65 ng
F-Fel d7	50 ng
dd H₂O	Up to 10 ul

Incubate samples in a thermocycler at 50°C for 15 minutes.

Following incubation, store samples on ice or at -20°C for subsequent transformation.

2.1.6 Transformation of E. coli DH5a

1.Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).

2. Mix 5 μl of assembled DNA into 50 μL of competent cells. GENTLY mix by flicking the bottom of the tube with your finger a few times.

3. Incubate the competent cell/DNA mixture on ice for 20-30 mins.

4. Heat shock each transformation tube by placing the tube into a 42°C water bath for 45 secs.

Put the tubes back on ice for 2 min.
Add 500 μl LB media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for more than 45 min.
Centrifuge to pellet the cells and decant most of the media.
Resuspend the cells and plate on the selection media according to the plasmid transformed.
Incubate plates at 37°C overnight.

2.1.7 Transformation of S. cerevisiae CEN. PK2-1C

Stock solution

1M LiAc
Materials	Usage	Note
LiAc-2H₂O	5.1 g/50 mL

50% PEG3350
Materials	Usage	Note
PEG3350	25 g/50 mL

Inoculate 5 mL of liquid YPD and incubate with shaking overnight at 30 °C .
Inoculate 0.5 mL overnight yeast cultured solution into 50 mL 2xYPD.
Incubate the culture at 30°C on a shaker at 200 rpm until OD600~0.5-1.2. This will take around 4-5 hours.
Harvest the culture in a sterile 50mL centrifuge tube at 4000 rpm for 5 min.
Pour off the medium, resuspend the cells in 25mL of sterile water and centrifuge again.
Pour off water, resuspend the cells in 25mL of sterile water and centrifuge again.
Pour off the water, resuspend the bacterial pellet from step (6) with X mL (i.e., the number of competent cell aliquots to be prepared) of sterile water.
Distribute the resuspended solution into X sterile 1.5 mL microcentrifuge tubes (1 mL per tube).
Centrifuge the tubes using a microcentrifuge at room temperature and 5000 rpm for 5 minutes.
After centrifugation, carefully discard the supernatant to obtain the yeast competent cell pellet.

11. Preparation of LiAc Mix

LiAc Mix Recipe (Single Aliquot)

LiAc Mix Recipe (each)
Materials	Usage	Note
50% PEG3350	260 μL
1 M LiAc	36 μL
Sterile Water	4 μL
2 mg/mL ssDNA	10 μL	Heat ssDNA at 95°for 5 min before use
dd H₂O	Up to 10 ul

For n aliquots: Prepare a mixture with a total volume of (n+2) times the single aliquot volume (i.e., scale up the single aliquot recipe by (n+2) times). After preparation, aliquot the mixture into (n+2) sterile 1.5 mL microcentrifuge tubes (including the required number for experimental and control groups, plus two extra aliquots as backups for potential loss).

(9) Preparation of DNA Mix

Prepare DNA Mix solutions for the experimental and control groups separately. Ensure that each DNA mix contains at least 250 ng of plasmid DNA, with a final volume of 50 μL.

(10) Chemical Transformation

a. Gently resuspend the competent cell pellet from step (7) with the corresponding DNA mix (50 μL) (Note: Competent cells must be used immediately after removal from the 4°C refrigerator).

b. After resuspension, add 310 μL of the LiAc mix from step (8) to each tube. Gently flick the tube or vortex to ensure thorough mixing, forming the Transformation Mix.

c. Precisely set a metal bath or water bath to 42°C. Place the Transformation Mix tubes from step b into the bath and heat-shock for 40 minutes.

d. After heat shock, centrifuge the tubes using a microcentrifuge at room temperature and 5000 rpm for 2 minutes. Carefully aspirate and discard the supernatant using a pipette, avoiding loss of the bacterial pellet at the bottom of the tube.

e. Add 500 μL of sterile water to each tube and gently pipette or vortex to resuspend the bacterial pellet. After resuspension, pipette 100 μL of the bacterial suspension and spread it evenly onto SC-Ura (Uracil-Deficient) selection plates.

f. Invert the spread plates and place them in a 30°C incubator to culture for 2-3 days. After cultivation, successfully transformed yeast monoclonal colonies should be visible.

2.2 The construction of ShRNA expression cassette

2.2.1 T4 PNK Phosphorylation

(1) Preparation of Oligo Pool

Mix 5 μL each of the upstream and downstream oligo DNA fragments. Vortex to mix thoroughly, then briefly centrifuge to collect the solution at the bottom of the tube to obtain the DNA Oligo Pool.

For example: CH1-shRNA1-F and CH1-shRNA1-R, CH1-shRNA2-F and CH1-shRNA2-R.

(2) T4 PNK Phosphorylation

Prepare the reaction mixture according to the table below, then place it in a PCR machine and run the reaction program shown in Figure 1.

T4 PNK Phosphorylation reaction mix (50 μL )

	50 μL
10 × T4 ligation buffer	5
DNA oligo pool	1
T4 Polynucleotide Kinase	1
dd H2O	43

T4 PNK Phosphorylation Reaction Program

Step	Programme	Notes
1	37°C，1 h	Phosphorylate oligo DNA ends with T4 PNK
2	98°C，5 min	Enzyme inactivation
3	10°C，∞

2.2.2 Digestion

	50μL
rCutSmart buffer	5
XbaI	3
BsaI-HFv2	3
pW1	1 μg
dd H2O	Up to 50 μL

Step	Programme	Notes
1	37°C，30 min
2	80°C，20 min	inactivation
3	10°C，∞

2.2.3 Extraction of Digestion Product

Refer to the instructions of the Universal DNA Purification Kit (GDP214). (Section 2.1.4 Gel extraction)

2.2.4 Ligation

	10 μL
10xT4 ligation Buffer	1
RD-pW1	60 ng
T4 PNK treated shRNAs oligo	5
T4 DNA ligase	0.5
dd H₂O	Up to 10 μL

Transformation of E. coli DH5α (see section 2.1.6 )

2.2.6 Sequencing

Since the fragment lengths of positive and negative colonies are similar, it is recommended to directly pick a single colony and culture it into liquid LB medium containing kanamycin overnight. On the next day morning, we extracted the plasmids following the procedures described in 2.1.1 Plasmid Extraction. Send to the sequencing facilities to get sequence information.

2.2.7 Transformation of E. coli HT115

Follow the instructions of the HT115 competent cell product.

(https://www.maokangbio.com/productView.action?id=9458)

2.2.8 Expanding Cultivation & IPTG Induction

(1) Inoculation

Pick a single colony of HT115 cells containing the transformed plasmid from the plate and inoculate it into a test tube containing liquid LB medium with kanamycin (K antibiotic). Shake at 37°C overnight to obtain the shRNA expression bacterial stock.

(2) Expanding Cultivation

Inoculate the bacterial stock at a 1:100 ratio into a 250 mL conical flask containing 50 mL of liquid LB medium with kanamycin. Shake at 37°C, 200–300 rpm, for 3–4 hours (4 hours) until OD600 reaches approximately 0.6.

(3) Induction

Before induction, take 1 mL of the sample as a pre-induction sample and store it at 4°C for subsequent RNA extraction and electrophoresis verification. Then add 1 M IPTG to a final concentration of 0.4 mM. Shake at 37°C for 4 hours.

(4) Bacterial Collection

Centrifuge the pre-induction sample and post-induction bacterial culture at 12,000 rpm for 2 minutes. Discard the supernatant and collect the bacterial pellet for RNA extraction.

2.2.9 RNA Extraction

We use RNAprep Pure Cell/Bacteria Kit (GDP-430) to extract RNAs.

1. Collect bacteria by centrifuging for 12,000 rpm (~13,400 × g) at 4°C in a centrifuge tube. Carefully remove all supernatant by aspiration, and proceed to step 2. The following steps should be completed at RT (15-30°C).

3. Re-suspend bacteria thoroughly with 100 μl TE buffer containing lysozyme (recipe and incubation time have been showed as follow).

G- bacterium 400 μg /ml 3-5 min

G+ bacterium 3 mg/ml 5-10 min

Add 350 μl Buffer RL (Ensure that β-Mercaptoethanol has been added to Buffer RL before use). Vortex or pipet to mix, and ensure that no cell clumps are visible before proceeding to step 5.
If insoluble precipitate has been formed, centrifuge for 2 min at 12,000 rpm (~13,400 × g). Transfer supernatant to another centrifuge tubes.

5. Add 250 μl ethanol to the cleared lysate, mix immediately by pipetting. Transfer the sample, including any precipitate that may have formed, to RNase-Free Spin Column CR3 placed in a 2 ml RNase-Free Collection Tube. Close the lid gently, and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the spin column back to the Collection Tube.

6. Add 350 μl Buffer RW1 to the RNase-Free Spin Column CR3. Close the lid gently, and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through.

7. Preparation of DNase I working solution: Add 10 μl DNase I stock solution (see Preparation of DNase I stock solution) to 70 μl Buffer RDD. Inverting the tube gently to mix.

8. Add the DNase I working solution (80 μl) directly to the center of RNaseFree Spin Column CR3, and place on the bench top for 15 min.

9. Add 350 μl Buffer RW1 to the RNase-Free Spin Column CR3. Close the lid gently, and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through.

10. Add 500 μl Buffer RW to RNase-Free Spin Column CR3 (Ensure that ethanol has been added to Buffer RW before use). Close the lid gently, place in room temperature for 2 min and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through.

11. Repeat step 10.

12. Centrifuge at 12,000 rpm (~ 13,400 × g) for 2 min, and then discard the waste liquid. Place the RNase-Free Spin Column CR3 at room temperature for 2-5 mins to completely dry the residual washing buffer in the column.

13. Place the RNase-Free Spin Column CR3 in a new 1.5 ml RNase-Free Collection Tube (supplied). Add 30-100 μl RNase-Free ddH2O directly to the spin column membrane. Close the lid gently, place at room temperature (15-30°C) for 2 min and then centrifuge for 2 min at 12,000 rpm (~13,400 × g) to elute the RNA.

14. Purified RNA is recommended be stored at -70°C.

2.2.10 RNA Electrophoresis

RNA electrophoresis can be performed using the common 1% Agarose-TAE gel method for DNA electrophoresis. Recommendations:

1. Clean the electrophoresis tank thoroughly before use, replace with fresh TAE electrophoresis buffer, and rinse related equipment with DEPC water before use.

2. If the RNA concentration meets the above standard of 100 μg/μL, directly take 2 μL RNA sample and mix with 10 μL 6× loading buffer. Load 3 μL of the mixture and run electrophoresis at 140 V for 20 minutes for verification.

3. After verification, store the extracted RNA samples at -20°C.

3.Validation of RNAi knockdown efficiency

3.1 RNA Chemical Transformation

1. Inoculate a reporter yeast colony into a test tube containing 5 mL SC-Ura liquid medium. Incubate at 30°C with shaking at 200 rpm overnight.

2. Inoculate the overnight reporter yeast culture at a 5% ratio into a 250 mL conical flask containing 50 mL of SC-Ura (-Uracil) liquid medium. Incubate at 30°C with shaking at 200 rpm until OD600 reaches ~0.5-0.8.

3.Centrifuge at 4,000 rpm, 20°C for 5 minutes, and discard the supernatant.

4.Resuspend the cell pellet in 1/2 volume of DEPC water. Centrifuge at 4,000 rpm, 20°C for 5 minutes to complete one wash cycle.

5.Repeat the washing step twice.

6.Take the yeast competent cells and resuspend them in the corresponding RNA mix. After resuspension, add 310 μL of RNA mix to each tube, mix well, and incubate in a 42°C water bath for 40 minutes.

RNA Mix Composition

LiAc Mix Recipe (each)
Materials	Usage	Note
50% PEG3350	260 μL
1 M LiAc	36 μL
RNA	~10 μL	(10 µg)
2 mg/mL ssDNA	10 μL	Heat ssDNA at 95°for 5 min before use
Nuclease free H₂O	Up to 310 ul

7.After the water bath incubation, centrifuge at 5,000 rpm for 2 minutes at room temperature and discard the supernatant.

8.Resuspend the cells in 500 μL of SC-Ura medium and transfer to a 2 mL culture tube.

9.Incubate at 30°C with shaking at 200 rpm for 16 hours for recovery.

3.2. Quantify fluorescence intensity

Measure fluorescence using a microplate reader (excitation wavelength: 488 nm, emission wavelength: 509 nm).

Step	Temperature (°C)	Time	Cycles
Initial Denaturation	95	5 min	1 cycle
Denaturation	95	30 s	Repeat steps 2-4 for 40 cycles
Annealing	56	30 s
Extension	72	X min (see table for details)
Final Extension	72	10 min	1 cycle
Hold	4	Indefinite	1 cycle

Microbial Media Preparation and Cultivation

E. Coli Media Preparation

Yeast Media Preparation

Molecular cloning

2.1 The construction of yeast reporter

2.1.1 Plasmid Extraction