1. Take competent cells out of -80°C and thaw them on the ice.
2. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and (optional) incubate them in 37°C incubator.
3. Mix 600ng of plasmids into 50μL of competent cells in a microcentrifuge tube. Gently mix them.
4. Incubate the competent cell and plasmid mixture on ice for 30 minutes.
5. Heat shock each transformation tube in a 42°C water bath for 90 seconds.
6. Put the tubes back on ice for 2 minutes.
7. Add 500μL of LB to the bacteria and grow them in a 37°C shaking incubator for 1 h.
8. Plate the transformation (about 150-200μL) onto a solid LB medium plate containing the appropriate antibiotic.
9. Incubate the plates at 37°C overnight.

I. Buffer preparation

1. Add the provided RNase A solution to Buffer I (use 1 vial RNase A per bottle Buffer I), mix, and store at 2-8°C.

II. Harvest bacterial precipitate

1. Take bacteria solution from the overnight culture (200mL LB in a conical flask with appropriate antibiotic) and transfer it into a 50mL centrifuge tube.
2. Centrifuge it at 5000 rpm for 10 minutes at 4℃ to get the bacterial precipitate. Try to remove all traces of supernatant, use clean paper tissue to absorb the fluids inside the tube wall.

III. Extraction of plasmid

1. Add 5mL of Buffer l to the bacterial centrifuge tubes, mix and shake the bacterial precipitate well to disperse it completely until no flocs exist. Transfer the bacterial suspension into a 50mL centrifuge tube.
2. Add 5mL of Buffer ll, gently invert the centrifuge tube 6-8 times, and leave it at room temperature for 5 minutes, so that the bacteria are completely lysed and the solution is clear.
3. Add 5mL of Buffer lll, immediately invert the centrifuge tube 6-8 times and mix well until white flocculent is produced. Leave on ice for 12 minutes.
4. Centrifuge the above lysate at 12,000g for 15minutes at 4°C. Carefully aspirate the supernatant and transfer to a new 50mL centrifuge tube.
5. Add 10mL of isopropanol. Mix well by inversion and leave on ice for 12 minutes.
6. Centrifuge at 12,000g for 10minutes at 4°C. Carefully discard the supernatant, invert and gently drain off the residual liquid, add 0.5mL of Buffer l to completely dissolve the precipitate clumps.
7. Transfer to a new 1.5mL centrifuge tube and leave at room temperature for 12 minutes.
8. Centrifuge the crude extract of plasmid at high speed for 2 minutes at room temperature in a desktop centrifuge, and transfer the supernatant into a new 1.5mL centrifuge tube.

IV. Plasmid purification

1. Add 100μL of Buffer IV (impurity removal solution A) to 0.5mL of plasmid crude extract, gently mix, 2,000g centrifugation for 2 minutes, the supernatant should be transferred to a new centrifuge tube.
2. Add 100μL of Buffer IV (Impurity Removal Solution A), mix gently, centrifuge at 12,000g for 5minutes and transfer the supernatant to a new tube.
3. Add 70μL of Buffer V (Impurity Removal Solution B), mix gently, centrifuge at 12,000g for 5 minutes, transfer the supernatant to a new centrifuge tube.
4. 0.5mL of isopropanol should be added, mixed well, and left at room temperature for 10 minutes. 12,000g centrifugation at room temperature for 10minutes, the supernatant should be discarded, washed gently with 1mL of 70% ethanol, the liquid should be discarded, and invert the tube. Cool it for 5 minutes at room temperature.
5. Dissolve precipitate with 0.5mL of ddH₂O.
6. Add 200μL of Buffer VI (Impurity Removal Solution C), mix well and leave on ice for 20 minutes, centrifuge at 12,000g for 10minutes at room temperature. Discard the supernatant, gently add 1mL of 70% ethanol to wash twice, invert and cool at room temperature for 5-10 minutes to make the ethanol evaporate completely.
7. Add appropriate amount of ddH₂O (200μL) to dissolve the precipitate (can be shaken in a 37°C water bath to aid dissolution).

1. Select restriction enzymes to digest plasmids: The amount of restriction enzyme is 1μL per 20μL system. (Detailed enzyme information followed https ://nebcloner.neb.com/ #! /redigest)
2. Calculate the system of restriction digest(20μL)

   Reagent	Amount
   Plasmid	600ng
   ddH2O	Appropriate volume
   Restriction enzyme	1μL of each enzyme
   rCutsmart	2μL

1. Measure the required amount of agarose.
2. Mix 1.2g of agarose powder with 140mL of 1×TAE buffer in a microwavable flask.
3. Microwave the mixture for 1 to 3 minutes until the agarose is completely dissolved.
4. Let agarose solution cool down to approximately 50°C.
5. Add the nucleic acid dye, usually about 20μL per 120mL of gel.
6. Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.
7. Place the newly poured gel at room temperature for 30-45 minutes until it has completely solidified.
8. Add loading buffer to each of your DNA samples.
9. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
10. Fill the gel box with 1×TAE buffer until the gel is covered.
11. Carefully load a marker into the first lane of the gel.
12. Carefully load your samples into the additional wells of the gel.
13. Run the gel at 135V until the dye line is approximately 75-80% of the way down the gel. The typical run time is about 45 minutes, which may vary depending on the gel concentration and voltage.
14. Turn OFF the power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
15. Using any device with UV light to visualize your DNA fragments. The DNA fragments are usually referred to as 'bands' due to their appearance on the gel.

1. Add the above reagents to a graduated container. Then, add RO water until the total volume reaches 1L.
2. Stir them at 660r/min on a magnetic stirrer, until the solutes are completely dissolved.
3. Aliquot the liquid into several conical flasks, 200ml each.
4. Sterilize the medium in an autoclave sterilizer for 20 minutes at a pressure of 15psi (1.05kg/cm²).

1. Add the reagents to graduate, and add RO water until the total volume reaches 1L.
2. Stir the mixture on a magnetic stirrer, until the solutes are completely dissolved.
3. Carefully transfer the prepared solution into the specialized bottle labeled for 50×TAE.
4. If 1×TAE solution is needed, add 20ml 50×TAE solution to 980ml ddH₂O

1. Preparation of Complete Adipogenic Induction Medium
   1. Thaw supplements and serum at room temperature.
   2. Prepare the induction medium according to experimental requirements.
2. Adipogenic Differentiation Induction
   1. Digest the cells, collect them by centrifuge, wash with PBS, and resuspend in complete medium supplemented with 10% FBS. Seed the cells evenly into well plates and culture in a 37°C constant temperature incubator.
   2. Begin induction when cell confluence reaches 80%-100%.
   3. Carefully aspirate the spent medium and slowly add pre-prepared induction medium along the well wall. Maintain cultures in the 37°C incubator. (Note: The medium must be pre-warmed to 37°C before adding to cells.)
   4. Replace with fresh induction medium every 2 days. Perform Oil Red O staining after approximately 14 days of induction.

1. Prepare the Oil Red O working solution by mixing the Oil Red O stock solution with distilled water at a 3:2 ratio and thoroughly vortexing the mixture. Filter the solution through filter paper and collect the filtrate, which is the ready-to-use Oil Red O working solution.
2. After completing the induction of differentiation, carefully aspirate the cell culture supernatant and wash the cells 1-2 times with 1×PBS. Add an appropriate amount of cell fixative and incubate at room temperature for 30 minutes for fixation.
3. Upon completion of cell fixation, carefully aspirate the fixative solution and wash the cells twice with 1×PBS.
4. Slowly add Oil Red O staining solution along the well wall, using 1mL per well, and incubate for 30 minutes at room temperature.
5. After staining, remove the staining solution and rinse with PBS to eliminate non-specific staining.

1. Seed cells in complete medium to reach 50-70% confluency at transfection. Incubate overnight (37°C, 5% CO₂).

1. Prepare transfection mix and incubate 5 min at RT.
   1. Tube A: Dilute plasmids in Opti-MEM (Take each well as an example)

      Reagent	Amount
      Opti-MEM	25μL
      Plasmids	According to the transfection system
      P3000 Regent	1μL

   2. Tube B: Dilute transfection reagent in Opti-MEM (Take each well as an example)

      Reagent	Amount
      Opti-MEM	25μL
      Lipofectamine 3000	1μL

2. Add tube A to tube B, mix gently.
3. Add the mixture to cells.
4. Swirl plate gently, and return it to incubator.

1. Incubate further (48h) for gene expression.

1. Thaw viral stock on ice.
2. Prepare infection mix.
3. Add infection mix to cells, mix well.
4. Incubate (37°C, 5% CO₂) for 24h and change the medium.
5. Using microscope to observe the mcherry.

1. Thaw CCK-8 solution and protect from light (wrap in foil).
2. Add 50μL CCK-8 reagent per well.
3. Return plate to CO₂ incubator for 2h.
4. The supernatant was then collected, transferred to a 96-well plate, and its absorbance was measured at 450nm using a microplate reader.

1. Remove the supernatant and wash the cells twice with PBS.
2. Lyse the cells by adding a total of 500μL of Transzol up per group. Collect the cell lysate.
3. Centrifuge the lysate at 12,000xg for 10 minutes at 4°C. After centrifugation, transfer the supernatant to a new tube.
4. Add 100μL of chloroform per tube, vortex for 15 seconds, and then shake for 30 seconds until the mixture turns a uniform pink color.
5. Incubate the mixture at room temperature for 5 minutes. And centrifuge the samples at 12,000xg for 15 minutes at 4°C.
6. Carefully transfer the upper to a fresh tube.Precipitate the RNA by adding 250μL of isopropyl alcohol pertube. Incubate at room temperature for 10 minutes.
7. Centrifuge at 12,000xg for 10 minutes at 4°C. Carefully discard the supernatant.
8. Wash the RNA pellet by adding 500ul of 75% ethanol Vortex briefly to dislodge the pellet.
9. Centrifuge at 7,500xg for 5 minutes at 4°C. Carefully aspirate the ethanol.
10. Air-dry the pellet for approximately 10 minutes and dissolve the purified RNA in an appropriate volume of nuclease-free water. Measure the concentration.

1. Prepare the reverse transcription mixture as follow

   Regents	Amount
   5x Reaction Buffer	4μL
   Enzyme mix	1μL
   ddH2O	5μL

2. Use ddH₂O to dilute 1000ng RNA extraction to 10μL
3. Adding the mixture and the RNA to the Thermal Cycler. Set the system at 50°C for 5min and 80°C for 0.05min. Waiting for the system temperature until it decrease to 4°C.
4. Adding 140μL ddH₂O to the tube. And remove the reverse transcription product to the 96 well PCR plate (4μL each)
5. Adding 5μL SYBR and 1μL primer, then seal the plate with a membrane.
6. Centrifuge at 2250 rpm for 3 minutes. Put the plate into the Real-Time PCR Instrument.

1. Conditioned medium collection
   1. Harvest MDA-MB-231 cells using trypsin-EDTA, centrifuge (1000 rpm, 5 min).
   2. Discard the supernatant and use PBS resuspend the cells.
   3. Centrifuge at 1000rpm for 5min, discard the supernatant and resuspend the cells in complete medium after washing by PBS.
   4. Seed breast cancer cells to 6 well plates and incubate at 37°C for 24h.
   5. Replace with 0.2% FBS-containing DMEM and cultivation for another 24 h.
   6. Collect the supernatant and filter using a 0.22 µm filter.
2. 1. Discard the old medium in the plate.
   2. Use MDA-MB-231 CM to wash cells once.
   3. Add 3ml MDA-MB-231 CM per well.
   4. Treat for 48h.

1. Move 50mL out of the unused DMEM medium (new bottle).
2. Add other reagents and stored the medium under 4°C.