Upload folder using huggingface_hub

DJI/DJI-Protocol/Add_cytokine_into_cells.txt

@@ -0,0 +1 @@
+Step1: Disinfect hands thoroughly with 70% ethanol.

DJI/DJI-Protocol/ColonyPCR.txt

@@ -0,0 +1,16 @@
+Colony PCR for 4 reactions
+1. Thaw all reagents
+2. Prepare a 10 μM primer mix. In a sterile 1.5 mL Eppendorf tube, add the following:
+a. 16 μL sterile H₂O
+b. 2 μL Forward Primer (from 100 μM stock)
+c. 2 μL Reverse Primer (from 100 μM stock)
+3. Prepare the PCR mix. In a new sterile 1.5 mL Eppendorf tube, combine the following:
+a. 24 μL sterile H₂O
+b. 6 μL of the primer mix (prepared above)
+c. 30 μL of 2x PCR Master Mix
+4. Aliquot 10 uL PCR mix into each well of 8-strip PCR tube.
+5. Using a 10 uL pipette tip, pick a single colony from the LB agar plate.
+6. Dip the pipette tip (with the picked colony) into a sterile 1.5 mL Eppendorf tube containing 200 μL of LB broth with the appropriate antibiotic.
+7. Without changing the tip, immediately dispense the pipette tip into the colony PCR mix in the 8-strip PCR tube
+8. after all colonies are picked, use channel pipette to gently dispense the colony and discard tips.
+8. Place the tube into the thermocycler.

DJI/DJI-Protocol/Demo_Cas9_delivery.txt

@@ -0,0 +1,8 @@
+1. Add 1 mL Opti-MEM into a sterile 1.5 mL EP tube.  
+2. Add Cas9 plasmid.  
+3. Add guide RNA plasmid.  
+4. Add PEI (4:1 ratio to DNA).  
+5. Incubate at room temperature for 20 min.  
+6. Add the mixture dropwise into a 10 cm dish of 293T cells (~70-80% confluency).  
+7. Gently rock the dish forward and backward to mix.  
+8. Place the dish back into the cell incubator (37°C, 5% CO₂).  

DJI/DJI-Protocol/Lentiviral_infection_of_iPSC-Fibroblast.txt

@@ -0,0 +1,13 @@
+1. Thaw purified Lenti-Cas12a-U6-TGFBR1 and control lentivirus on ice.
+
+2. Retrieve pre-seeded iPSC-fibroblast plates from the incubator.
+
+3. (Optional) Replace culture medium with fresh infection medium as needed.
+
+3. Add 20 µL of the appropriate virus to each well.
+
+4. Gently rock the plate to evenly distribute the virus.
+
+5. Record the infection start time and group assignments on the plate and in your log.
+
+6. Return plates to the incubator and culture for 72 hours.

DJI/DJI-Protocol/Lentiviral_infection_of_iPSCs_XW.txt

@@ -0,0 +1,7 @@
+Protocol: Lentiviral Infection of Target Cells
+1.	Seed target cells in a 10 cm dish one day prior to infection to reach ~50% confluency.
+2.	Thaw the required aliquot of lentivirus on ice.
+3.	Add polybrene to the culture medium at a final concentration of 8 µg/mL.
+4.	Add the lentivirus suspension to the cells at the desired MOI (multiplicity of infection).
+5.	Gently swirl the plate to ensure even distribution.
+6.	Incubate the cells under standard culture conditions (37°C, 5% CO₂) overnight.

DJI/DJI-Protocol/Loading_DNA_samples_on_E-gel.txt

@@ -0,0 +1 @@
+Protocol: Loading DNA Samples on E-gel

DJI/DJI-Protocol/Mock_Cas9_Delivery.txt

@@ -0,0 +1,7 @@
+1. Add reagent 1 into a sterile 1.5 mL EP tube.  
+2. Add  reagent 2 into the EP tube.  
+3. Add  reagent 3 into the EP tube. 
+4. Mix well. 
+5. Incubate at room temperature for 20 min.  
+6. Add the mixture dropwise into a 10 cm dish of 293T cells (~70-80% confluency).  
+7. Gently rock the dish forward and backward to mix.  

DJI/DJI-Protocol/Mock_Transformation.txt

@@ -0,0 +1,7 @@
+1. Add 5 μL of plasmid DNA to the competent E. coli cells.
+2. Mix gently by flicking the tube 4-5 times. 
+3. Incubate the DNA-cell mixture on ice for 5 seconds.
+4. Rapidly transfer the tube to a pre-heated 42°C water bath for exactly 5 seconds.
+5. Immediately return the tube to ice for  5 seconds.
+6. Spread 50μL of culture onto an LB agar plate containing the appropriate selective antibiotic.
+7. Invert the plates. 

DJI/DJI-Protocol/PCR_Reaction_Setup.txt

@@ -0,0 +1,15 @@
+Protocol: PCR Reaction Setup
+1. 	Thaw all reagents (Master Mix, primers, DNA template, nuclease-free water) and keep them on ice.
+2. 	In a sterile 0.2 mL PCR tube, add 12.5 μL of 2x Master Mix.
+3. 	Add 1 μL of Forward Primer (10 μM stock).
+4. 	Add 1 μL of Reverse Primer (10 μM stock).
+5. 	Add 2 μL of template DNA (concentration approx. 20 ng/μL).
+6. 	Add 8.5 μL of nuclease-free water to reach a final volume of 25 μL.
+7. 	Mix contents by gently flicking the tube, then perform a quick spin in a microcentrifuge to collect the liquid at the bottom.
+8. 	Place the tube into the thermocycler.
+9. Program the cycler or load pre-configured program:
+o Initial denaturation at 95°C for 3 minutes
+o 30 cycles of: 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min
+o Final extension at 72°C for 5 minutes
+10. Start the program.
+

DJI/DJI-Protocol/Passaging&Seeding_293T_Cells.txt

@@ -0,0 +1,11 @@
+1. Aspirate the old medium from the 10 cm dish.  
+2. Wash cells with 5 mL PBS and aspirate completely.  
+3. Add 1 mL trypsin-EDTA and incubate at 37°C for 2 minutes.  
+4. Tap the dish to detach cells, then add 4 mL complete medium to neutralize trypsin.  
+5. Pipette the suspension 5-10 times to break cell clumps.  
+6. Take 100 μL cell suspension, mix with 100 μL trypan blue, and count cells using a hemocytometer.  
+7. Calculate the volume needed for 0.1 million cells per well in the 24-well plate.  
+8. Add 500 μL DMEM to each well first, then add the calculated cell volume.  
+9. Gently tap the plate edges to mix cells evenly.  
+10. Transfer 1/8 of the remaining suspension to a new 10 cm dish with 10 mL fresh medium.  
+11. Incubate both the 24-well plate and 10 cm dish at 37°C with 5% CO₂.

DJI/DJI-Protocol/Splitting cells_DJI-025.txt

@@ -0,0 +1,24 @@
+1. Spray hands with 70% ethanol 
+2. Get all reagents 
+3. Spray all materials with 70% ethanol before placing in hood
+4. Remove 10cm dish from incubator
+5. Place dish in biosafety cabinet
+6. Aspirate old medium completely using vacuum aspirator
+7. Tilt dish to remove residual medium from edges
+8. Add 3ml PBS to dish
+9. Gently rock dish to wash cell surface
+10. Aspirate PBS completely
+11. Add 1ml of 0.25% Trypsin-EDTA to 10cm dish
+12. Ensure even distribution by tilting dish
+13. Incubate at 37°C for 1 minutes
+14. Gently tap sides of dish to help detachment
+15. Add 5ml complete medium to dish
+16. Pipette up and down 5-10 times to create single cell suspension
+17. Transfer entire 6ml cell suspension to sterile 50ml conical tube
+18. Calculate volume needed for 1:3 split (approximately 2ml of cell suspension)
+19. Add 5ml cell suspension to new 10cm dish
+20. Add 5ml fresh medium (total 10ml in dish)
+21. Gently rock dish in cross pattern (North-South, East-West) to distribute cells
+22. Place dishes back
+23. Ensure dishes are level, clean-up, close hood
+

DJI/DJI-Protocol/Splitting cells_DJI-026.txt

@@ -0,0 +1,22 @@
+1. Spray hands with 70% ethanol 
+2. Get all reagents 
+3. Spray all materials with 70% ethanol before placing in hood
+4. Remove 10cm dish from incubator
+5. Place dish in biosafety cabinet
+6. Aspirate old medium completely using vacuum aspirator
+7. Tilt dish to remove residual medium from edges
+8. Add 1ml of 0.25% Trypsin-EDTA to 10cm dish
+9. Ensure even distribution by tilting dish
+10. Incubate at 37°C for 1 minutes
+11. Gently tap sides of dish to help detachment
+12. Add 5ml complete medium to dish
+13. Pipette up and down 5-10 times to create single cell suspension
+14. Transfer entire 6ml cell suspension to sterile 50ml conical tube
+15. Calculate volume needed for 1:3 split (approximately 2ml of cell suspension)
+16. Add 5ml cell suspension to new 10cm dish
+17. Add 5ml fresh medium (total 10ml in dish)
+18. Gently rock dish in cross pattern (North-South, East-West) to distribute cells
+19. Place dishes back
+20. Ensure dishes are level, clean-up, close hood
+
+

DJI/DJI-Protocol/Splitting cells_DJI-027.txt

@@ -0,0 +1 @@
+1. Spray hands with 70% ethanol 

DJI/DJI-Protocol/Virus_collecting_XW.txt

@@ -0,0 +1,4 @@
+Virus_collecting
+1.	 Prepare 15 ml centrifuge tube, 6 ml syringe and 0.45 um filter.
+2.	Carefully collect the viral supernatant from the producer cells and pass it through the 0.45 µm syringe filter into the 15 mL centrifuge tube to remove cell debris.
+3.	Dispense the filtered viral solution into 1.5 mL microcentrifuge tubes.

DJI/DJI-Protocol/Virus_packging_tranfection_of_293T_cells_XW.txt

@@ -0,0 +1,10 @@
+Virus_packging_tranfection_of_293T_cells
+1.	Take HEK293T cells and culture them to ~70% confluency in a 10 cm dish.
+2.	In a sterile 1.5 mL tube, mix 10 µg lentiviral backbone plasmid, 7.5 µg packaging plasmid, and 5 µg envelope plasmid.
+3.	Add 60 µL of transfection reagent and bring the volume up to 300 µL with serum-free medium.
+4.	Incubate the mixture at room temperature for 15 minutes.
+5.	Add the transfection mi x dropwise to the HEK293T cells.
+6.	Gently swirl the dish to evenly distribute the complex.
+7.	Incubate cells for 48–72 hours and harvest the viral supernatant.
+
+

DJI/DJI-Protocol/iPSC-Fibroblast Harvest and Lysis with QuickExtract.txt

@@ -0,0 +1,9 @@
+	1	haw purified Lenti-Cas12a-U6-TGFBR1 and control lentivirus on ice.
+	2	Retrieve pre-seeded iPSC-fibroblast plates from the incubator.
+	3	(Optional) Replace culture medium with fresh infection medium as needed.
+	4	Add 20 µL of the appropriate virus to each well.
+	5	Gently rock the plate to evenly distribute the virus.
+	6	Record the infection start time and group assignments on the plate.
+	7	Return plates to the incubator and culture for 72 hours.
+
+