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XMglass/XMprotocol/ColonyPCR.txt

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+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.

XMglass/XMprotocol/Freeze_Cell_1_YiZhu.txt

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+1) Harvest cells at ~80% confluency by trypsinization or scraping as appropriate.
+
+

XMglass/XMprotocol/Freeze_Cell_2_YiZhu.txt

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+2) Transfer the cell suspension into a 15 mL conical tube.
+3) Centrifuge at 300 × g for 5 minutes at room temperature.

XMglass/XMprotocol/Freeze_Cell_3_YiZhu.txt

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+4) Aspirate the supernatant and gently resuspend the pellet in freezing medium (90% FBS + 10% DMSO).
+5) Aliquot 1 mL of cell suspension into sterile cryovials.

XMglass/XMprotocol/Lentiviral_infection_of_iPSC-Fibroblast.txt

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+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.

XMglass/XMprotocol/Lentiviral_infection_of_iPSCs.txt

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+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.

XMglass/XMprotocol/Loading_DNA_sample_on_E-gel.txt

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+1. Place the Invitrogen E-gel cassette into the E-gel power base.
+2. Load 10 µL of the prepared DNA sample into a well.
+3. Add 10 µL of DNA ladder into a separate well.
+4. Press the “Run” button on the E-gel power base to start electrophoresis.

XMglass/XMprotocol/Loading_DNA_sample_on_E-gel_XW.txt

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+Protocol: Loading DNA Samples on E-gel

XMglass/XMprotocol/Mock_data_PCR.zip

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+version https://git-lfs.github.com/spec/v1
+oid sha256:688bd44e29261099f52f9abc5282ae162bbe9cb5fbd363bc081fc42ebed36017
+size 18487

XMglass/XMprotocol/PCR Reaction Setup for sanger and TIDE analysis.txt

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+1. Retrieve lysed iPSC-fibroblast samples from the thermal cycler and place on ice.
+
+2. Thaw PCR mix buffer and primers on ice, then briefly vortex and spin down.
+
+3. For each reaction, add 10 µL PCR mix buffer to the PCR tube/well.
+
+4. Add 1 µL forward primer (10 nM) and 1 µL reverse primer (10 nM) to each reaction.
+
+5. Add 2 µL QuickExtract lysate as the DNA template.
+
+6. Add 8 µL nuclease-free water (ddH₂O) to bring the total volume to 22 µL.
+
+7. Gently mix, briefly spin down to collect contents, and seal the tubes/plate.
+
+8. Place reactions into the PCR machine and select the saved Phanta PCR amplification program.
+
+9. Start the run and proceed to downstream analysis upon completion.

XMglass/XMprotocol/PCR_Reaction_Setup.txt

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+1. In a sterile 0.2 mL PCR tube, add Master Mix (in the tube labeled M).
+2. Add Forward Primer (in the tube labeled F).
+3. Add Reverse Primer (in the tube labeled R).
+4. Add template DNA (in the tube labeled T).
+5. Mix contents by gently flicking the tube, then perform a quick spin in a microcentrifuge to collect the liquid at the bottom.
+6. Place the tube into the thermocycler.

XMglass/XMprotocol/PCR_Reaction_Setup_XW.txt

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+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.
+

XMglass/XMprotocol/RNA_Extraction_YiZhu.txt

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+1) Aspirate and discard the culture medium from the cells.
+2) Wash the cells once with 1 mL sterile PBS to remove residual medium.
+3) Add 500 µL of RNA lysis buffer directly onto the cells in the dish.
+4) Pipette up and down 5–10 times to lyse and homogenize the cells completely.
+5) Transfer the lysate into a sterile RNase-free 1.5 mL microcentrifuge tube.
+6) Add an equal volume of 70% ethanol to the lysate and mix gently by pipetting.

XMglass/XMprotocol/Raw_Data_YiZhu.zip

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+version https://git-lfs.github.com/spec/v1
+oid sha256:4e3f27af25a0738e4e7371a563158864945ae6c8f9c3529d11607a7ed8334d33
+size 1003617

XMglass/XMprotocol/Serial_Dilution.txt

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+1. Label four sterile 0.2 mL PCR tubes as 1, 2, 3 and 4.
+2. Add 100 µL of sterile water (in the tube labeled H2O) to each tube.
+3. Add 100 µL of the original sample (in the tube labeled S) into the tube 1 and mix well by pipetting.
+4. Transfer 100 µL from the tube 1 into the tube 2, mix well by pipetting.
+5. Transfer 100 µL from the tube 2 into the tube 3, mix well by pipetting.
+6. Transfer 100 µL from the tube 3 into the tube 4, mix well by pipetting.
+

XMglass/XMprotocol/Serial_Dilution_YiZhu.txt

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+1) Mark four sterile PCR tubes as 1, 10⁻¹ 10⁻² and 10⁻³.
+2) Add 90 µL of original sample to the first tube. Add 90 µL of buffer to rest tubes.
+3) Add 10 µL of the original sample into the 10⁻¹ tube and mix well by pipetting.
+4) Transfer 10 µL from the 10⁻¹ tube into the 10⁻² tube, mix well. Repeat the process sequentially for 10⁻³ tubes.
+

XMglass/XMprotocol/Virus_collecting_XW.txt

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+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.

XMglass/XMprotocol/Virus_packging_tranfection_of_293T_cells_XW.txt

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+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.
+
+

XMglass/XMprotocol/iPSC-Fibroblast Harvest and Lysis with QuickExtract.txt

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+	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.
+
+

XMglass/XMprotocol/lentivirus_infection_YiZhu.txt

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+1) Seed target cells in a 6cm dish one day prior to infection to reach ~50% confluency.
+2) Add polybrene to the culture medium at a final concentration of 8 µg/mL.
+3) Add the lentivirus suspension to the cells at the desired MOI (multiplicity of infection).
+Gently swirl the plate to ensure even distribution.
+4) Incubate the cells under standard culture conditions (37°C, 5% CO₂) overnight.