Methods #

sgRNA Barcode Library Plasmid Pool Assembly #

1. Perform an extension reaction to generate double-stranded insert gRNA barcode DNA. Mix 10 $\mu$L NEB 5X Q5 Reaction Buffer, 1 $\mu$L of 10 mM dNTPs, 2 $\mu$L 100$\mu$M CROPseq-PrimeF-BgL-BsmBI, 1 $\mu$L 100$\mu$M CROPseq-RevExt-BgL-BsmBI , and 0.5 $\mu$L to create a 50 $\mu$L reaction.¹ See 10X-3.1.1 for 10X vector variation.

2. Run the extension reaction on a thermocycler using the following settings: (1) 98 °C for 2 min, (2) 65 °C for 30 sec, (3) 72 °C for 10 sec, (4) Repeat steps 2-3 for 10 cycles, (5) 72 °C for 1 min, (6) 4 °C hold

3. Confirm dsDNA assembly on 2% agarose gel by running single stranded DNA against PCR product.

4. Digest 5-10 $\mu$g of CROPseq vector backbone in a reaction containing 20 $\mu$L Digestion Buffer 3.1, 8 $\mu$L BsmBI, and nuclease-fee water to 200 $\mu$L. See 10X-3.1.4 for 10X vector variation.

5. Run the digested backbone on a 1-1.5% low melting point agarose gel, then follow the instructions on a DNA gel purification kit to extract and purify the linearized plasmid band.

6. Ligate double stranded gRNA barcode DNA into linearized gRNA transfer vector at a molar ratio of 10:1 in a 50X Golden Gate assembly reaction by mixing 1.25 pmol digested gRNA transfer vector (from step 3.1.4), 12.5 pmol double stranded gRNA barcode DNA (from step 3.1.1), 50 $\mu$L T4 ligase buffer, 25 $\mu$L T7 ligase, 25 $\mu$L BsmBI (CROPseq) or BbsI (10X Capture), and nuclease-free water to 500 $\mu$L.²

7. Run the Golden Gate assembly reaction on a thermocycler overnight using the following settings: (1) 42 °C for 2 min, (2) 16 °C for 5 min, (3) Repeat steps 1-2 for 99 cycles, (4) 55 °C for 30 min, (6) 4 °C hold

8. Clean barcoding library plasmid pool using a DNA clean and concentrator kit and elute in 22 $\mu$L warm, nuclease-free water.³

9. Prepare for e. coli electroporation by pre-warming recovery media to room temperature, thawing electrocompetent e. coli on ice, and pre-chilling 2 mm electroporation cuvettes on ice.⁴

10. Aliquot 100 $\mu$L of E.coli into the chilled 0.2 cm electroporation cuvette, add 5 $\mu$L of purified assembled plasmid, and stir with pipet tip.⁵

11. Transform e. coli by electroporating with 1 pulse at 2.5 kV.⁶

12. Add 2 mL Recovery Media and gently pipet up and down immediately after electroporation, and transfer to a sterile 50ml conical tube.

13. Repeat steps 10-12 three times

14. Allow cells to recover for 30 min at 37 °C with shaking at 250 rpm.

15. Pre-warm 2xYT agar plates with 100 $\mu$g/mL carbenicillin.

16. After recovery, perform dilution plating 1:10⁴, 1:10⁵, 1:10⁶ on carbenicillin agar plates.

17. Incubate plates overnight at 37 °C.

18. Put the remaining transformant mixture into 500 mL 2xYT with 100 $\mu$g/mL carbenicillin in a 2 L flasks.

19. Incubate flasks at 30 °C overnight with shaking at 250 rpm.

20. The culture can be pelleted or midi/maxi prepped for usage.

21. Calculate transformation efficiency from dilution plating.⁷

sgRNA Barcode Sampling #

The diversity of the initial plasmid pool should be assessed to ensure a high diversity library. To do this, PCR is performed with primers containing Illumina indices that anneal to regions flanking the barcodes.

1. Midi-prep one tube of transformed e. coli from step 3.1.1 according to manufacturer’s instructions.

2. Create the phasing primer mixture ‘PrimeF-PAS’ by mixing equimolar amounts of PrimeF-PASx0, PrimeF-PASx1, PrimeF-PASx2, PrimeF-PASx3, PrimeF-PASx4, and PrimeF-PASx5 (see Table 1).⁸

3. Prepare the PCR reaction to amplify barcodes and add Illumina indices by mixing 10 uL 5X Q5 Reaction Buffer, 1 $\mu$L 10 mM dNTPs, 2.5 $\mu$L PrimeF-PAS (from step 3.2.2), 2.5 $\mu$L PrimeR_cropseq (see Table 1), 0.5 $\mu$L Q5 polymerase, 50 ng plasmid DNA, and nuclease-free water to 50 $\mu$L. ⁹ See 10X-3.2.3 for 10X vector variation.

4. Amplify the barcodes by running the 50 $\mu$L reaction on a thermocycler using the following settings¹⁰: (1) 98°C for 5 min, (2) 98°C for 10 sec, (3) 63°C for 30 sec, (4) 72°C for 15 sec, (4) Repeat steps 2-4 for 7 cycles¹¹, (5) 72°C for 2 min, (6) 15°C hold

5. Transfer 50 $\mu$L PCR amplification product to a nuclease-free microcentrifuge tube

6. Allow SPRI beads to come to room temperature.

7. Add 35 $\mu$L (0.7X) paramagnetic SPRI beads and mix well with vortexing or pipetting up and down 10 times.

8. Incubate at room temperature for 5 minutes.

9. Place the tube on a magnetic rack and allow solution to clear (5-10 minutes).

10. While the tube is on the rack transfer the clear supernatant to a new tube without disturbing the bead pellet.

11. Add 55 $\mu$L (1.8-0.7x) paramagnetic SPRI beads to the supernatant from step 10 and mix well with vortexing or pipetting up and down 10 times.

12. Place the tube on a magnetic rack and allow solution to clear (5-10 minutes).

13. With the tube still in the rack, aspirate the clear supernatant.

14. With the tube still in the rack, add 180 $\mu$L of 80% ethanol and allow it to sit for 30 seconds.¹²

15. With the tube still in the rack, aspirate the ethanol and repeat step 14.

16. Remove supernatant and allow bead to dry for no more than 5 minutes.¹³

17. Remove tube from the magnetic rack and elute DNA by adding 42 $\mu$L of nuclease-free water.

18. Incubate at room temperature for 10 minutes.

19. Transfer tube to magnetic rack and collect 40 $\mu$L of purified PCR product after solution has cleared (5-10 minutes).¹⁴

18. Quantify DNA yield with a high sensitivity fluorometry kit ensuring yield between 0.5-10 ng/$\mu$L.

19. Load sample on to BioAnalyzer chip according to the manufacturer’s protocol and ensure a clear peak around 225 bp.¹⁵

20. Submit sample for Illumina sequencing.

21. See 3.7.2 for processing barcode sequence data.

SgRNA Barcoding Lentivirus Production #

1. 48 hours before transfection, plate 0.22-0.25 x 10⁶ low-passage HEK-293T cells in DMEM supplemented with 10% FBS without antibiotics in each well of a sterile 6-well tissue culture treated plate such that cells will be 70-80% confluent at the time of transfection.

2. On the morning of transfection, replace media on HEK-293T cells with 2 mL of fresh Opti-MEM^TM (or your cells growth medium) supplemented with 10% FBS without antibiotics.

3. In the afternoon, warm Opti-MEM^TM, Lipofectamine^TM 2000, and VSV-G, psPAX, and sgRNA barcoding plasmid to room temperature.¹⁶¹⁷

4. Per well of a 6 well plate, prepare “Tube A” containing 150 $\mu$L Opti-MEM^TM and 9 $\mu$L Lipofectamine^TM 2000.¹⁸

5. Incubate “Tube A” at room temperature for 5 minutes.

6. Per well of a 6 well plate, prepare “Tube B” containing 150 $\mu$L Opti-MEM^TM,1.5 $\mu$g psPax, 0.4 $\mu$g VSV-G, 3-5 $\mu$g sgRNA barcoding plasmid.

7. Slowly add “Tube B” dropwise to “Tube A” and carefully mix by gently inverting 10 times

8. Incubate at room temperature for 20 minutes.

9. Add 300 $\mu$L of the transfection mix slowly and dropwise to each well of HEK-293T cells.

10. 16-18 hours post-transfection, carefully remove and dipose of media containing Lipofectamine^TM 2000 complexes and slowly replenish with DMEM supplemented with 20% FBS without antibiotics.¹⁹²⁰

11. 48 hours post-transfection, harvest viral containing supernatant and store in a 50 mL conical tube at 4 °C. Optional: To increase viral harvest,replenish media dropwise on virus producing cells for next day collection. ²¹

12. Optional: 72 hours post-transfection, harvest final viral containing supernatant and combine with 48 hour viral collection.²²²³

13. Spin down collected viral containing supernatant at 500 x g for 10 min at 4 °C to remove residual HEK-293T cells.

14. Remove plunger from 20 mL syringe and attach to a 0.45 $\mu$m PES syringe filter.

15. Transfer viral supernatant to the 20 mL syringe.

16. Filter viral supernatant through 0.45 $\mu$m PES syringe filter into a fresh 50 mL conical tube to remove any remaining cell debris.

17. Concentrate virus ~20X in 30,000 MWCO PES ultrafiltration centrifugal concentrator by loading 20 mL of filtered viral supernatant into concentrator chamber and spinning at 4000 x g for 60-75 minutes at 4 °C until ~1 mL of media remains in filter.²⁴

18. Aliquot 25-50 $\mu$L of concentrated virus in threaded cryovials and store at -80 °C.²⁵²⁶

Determine sgRNA Viral Titer #

²⁷²⁸

Titering on Adherent Cells (Forward Procedure) #

²⁹

1. 24-48 hours before performing viral transduction seed your cell line of interest in a 12-well plate such that it is near 60-70% confluent at time of transduction.

2. Prior to transduction, one well of the replicate 12 wells should be dissociated and counted using trypan blue exclusion on a hemocytometer to know approximate number of live cells at time of transduction.³⁰³¹

3. Create stock of media containing your cells’ standard growth medium supplemented with 20% FBS containing 0-10 $\mu$g/mL hexadimethrine bromide (1:1000 dilution from hexadimethrine bromide stock to get 10 $\mu$g/mL).³²

4. Place 600 $\mu$L of hexadimethrine bromide containing medium separate microcentrifuge tubes.

5. Add virus in increasing amounts to each tube.

7. Incubate for 16 hrs at 37 °C, then carefully remove viral containing supernatant and replace with complete growth medium.³³³⁴

8. Incubate for an additional 32 hrs at 37 °C, then remove medium and wash each well gently with PBS.³⁵

9. Dissociate the cells from the plate and centrifuge at 300 x g for 5 minutes at 4 °C.

10. Wash cell pellets with PBS and repeat spin.

    1. Perform this step three times to ensure removal of trace virus before flow cytometry.

11. Resuspend cells in chilled FACS Buffer.³⁶

12. Keep cells on ice and continue to step 3.4.3

Titering on Suspension Cells #

1. Count your cells of interest using a hemocytometer.

2. Create stock of media containing your cells’ standard growth medium supplemented with 20% FBS containing 0-10 $\mu$g/mL hexadimethrine bromide (1:1000 dilution from hexadimethrine bromide stock for 10 $\mu$g/mL).³⁷.

3. Resuspend 1.20 x 10⁶ cells in 7.2 mL of containing hexadimethrine bromide media such that the final solution contains 1 x 10⁵ cells in 600 $\mu$L.

4. Plate 600 $\mu$L of cell solution in 10 wells of a tissue culture treated 12-well plate

5. Add virus in increasing amounts to each well and mix well.³⁸

6. Incubate for 16 hrs at 37 °C.³⁹⁴⁰

7. Transfer cell suspensions to sterile 1.7 mL microcentrifuge tubes and spin down at 500 x g for 5 minutes at 4 °C.⁴¹

8. Resuspend each cell pellet with complete growth medium and transfer to fresh 12-well plate.

9. Incubate for an additional 32 hrs at 37 °C, then transfer wells to microcentrifuge tubes and spin down at 400 x g for 5 minutes at 4 °C.⁴²

10. Wash cell pellets with PBS and repeat spin.⁴³

11. Resuspend cells in chilled FACS Buffer.⁴⁴

12. Keep cells on ice and continue to step 3.4.3

Flow Cytometry to Determine Viral Titer #

1. Pass cells resuspended in FACS buffer through a 35 $\mu$m nylon mesh strainer into a 5 mL flow cytometry test tube.⁴⁵

2. Use control samples to set laser voltages on FSC-A, SSC-A, and BFP such that nearly all cells are seen within FSC-A vs. SSC-A plot and both negative and positive populations can be seen and distinguished on the BFP channel.⁴⁶

3. After setting voltages with control samples, run transduced samples from lowest viral to highest. Set the cytometer to record at least 10,000 events for each sample. Record %BFP-positive for each titration.

4. Create a plot showing volume of virus on the x-axis and %BFP-positive on the y-axis.⁴⁷

Calculate viral titer in titering units (TU) per mL using Equation 1 using a pair of values within the linear region of the titer curve.⁴⁸

$$\frac{\text{TU}}{\text{mL}}\text{=}\frac{\left(\text{Number of cells at time of transduction} \right)\text{ × }\left( \text{Fraction of Positive Cells} \right)}{\left( \text{Volume of virus }\left\lbrack \text{mL} \right\rbrack \right)}$$

sgRNA Barcode Transduction #

1. After calculating the viral titer (TU/mL) on your cell line of interest, determine the final number of cells you require for your experiment using and transduce cells at a multiplicity of infection (MOI) of 0.1 (Equation 2) to minimize the occurrence of multiple barcode integrations.⁴⁹⁵⁰

2. Use control samples to set laser voltages on FSC-A, SSC-A, and BFP such that nearly all cells are seen within FSC-A vs. SSC-A plot and both negative and positive .populations can be seen and distinguished on the BFP channel.⁵¹

3. Set sort gate on BFP-positive cells indicative of a productive sgRNA barcode.⁵²

4. Sort cells on BFP-positive gate via FACS.

5. Maintain sorted cells in culture with complete growth medium.

$$\text{MOI [TU/cell] = }\frac{\left( \text{Volume of Virus needed [mL]} \right)\text{ × }\left( \text{Titer of Virus [TU/mL]} \right)}{\left( \text{Number of cells exposed to virus} \right)}\text{ = 0.1}$$

Targeted sgRNA Barcode Sampling of Cells #

Preparing Samples for Sequencing #

1. To assess cell barcode diversity harvest cells from culture and collect into cell pellet.⁵³

2. Isolate genomic DNA from cell pellet using kit or standard protocol and proceed to PCR amplification.

3. Prepare the PCR reaction to amplify barcodes and add Illumina indices⁵⁴ by mixing 10 uL 5X Q5 Reaction Buffer, 1 $\mu$L 10 mM dNTPs, 2.5 $\mu$L PrimeF-PAS (from step 3.2.2), 2.5 $\mu$L PrimeR_cropseq (see Table 1), 0.5 $\mu$L Q5 polymerase, up to 2 $\mu$g genomic DNA⁵⁵, and nuclease-free water to 50 $\mu$L. See 10X-3.6.1.3 for 10X vector variation.

4. Amplify the barcodes by running the 50 $\mu$L reaction on a thermocycler using the following settings⁵⁶: (1) 98°C for 5 min, (2) 98°C for 10 sec, (3) 63°C for 30 sec, (4) 72°C for 15 sec, (4) Repeat steps 2-4 for 25 cycles⁵⁷, (5) 72°C for 2 min, (6) 15°C hold

5. Finish preparing barcodes as described in 3.2 steps 4-20.

Processing Barcode Sequencing Data (WIP!) #

See pycashier for more info about how to get started processing fastq data to get barcode information.

Recall Plasmid Assembly #

1. 3 pairs of overlapping oligos containing the barcode sequence of interest flanked by overlapping sequences should be ordered according to Table 1.⁵⁸

2. In separate tubes, mix each of the 100 $\mu$M oligo pairs together:

• Tube AB: 10 $\mu$L Bg-AB-fwd + 10 $\mu$L Bg-AB-rev
• Tube BC: 10 $\mu$L Bg-BC-fwd + 10 $\mu$L Bg-BC-rev
• Tube CD: 10 $\mu$L Bg-CD-fwd + 10 $\mu$L Bg-CD-rev

3. Heat each to 80 °C and let cool to create DNA blocks containing a barcode, a PAM site, and overhang sequences.⁵⁹

4. Ligate DNA blocks together creating the barcode array by mixing 10 $\mu$L “Tube AB”, 10 $\mu$L “Tube BC”, 10 $\mu$L “Tube CD”, 5 $\mu$L 10 mM dNTPs, 5 $\mu$L 10X T4 PNK buffer, 1 $\mu$L T4 PNK, and 9 $\mu$L nuclease-free water (50 $\mu$L reaction volume).

5. Incubate at 37 °C for 45 minutes.

6. Add 2 $\mu$L T7 DNA ligase to the 50 $\mu$L mixture and incubate at room temperature overnight.

7. Run ligation product in a 2% agarose gel and gel purify band from approximately 170 bp.

8. Ligate the barcode array into the recall plasmid backbone at a molar ratio of 10:1 in a Golden Gate assembly reaction by mixing 25 fmol Recall-miniCMV-sfGFP, 250 fmol assembled barcode array (from step 3.8.5), 1 $\mu$L T4 ligase buffer, 0.5 $\mu$L T7 ligase, 0.5 $\mu$L BbsI, and nuclease-free water to 10 $\mu$L.

9. Run the Golden Gate assembly reaction on a thermocycler using the following settings: (1) 42 °C for 2 min, (2) 16 °C for 5 min, (3) Repeat steps 1-2 for 35 cycles, (4) 55 °C for 30 min, (6) 4 °C hold

10. Transform bacteria with golden gate product.⁶⁰

11. Verify insertion of barcode array into Recall-miniCMV-sfGFP backbone via Sanger sequencing.

Recall and Isolation of Barcoded Lineages #

⁶¹⁶²

1. 24-48 hours before performing recall transfection seed your cell line of interest in growth medium in a 6-well plate such that it is near 60-80% confluent at time of transfection.

2. Per well of a 6 well plate, prepare “Tube A” containing 100 $\mu$L Opti-MEM^TM and 9 $\mu$L Lipofectamine^TM 3000.⁶³

3. Incubate “Tube A” at room temperature for 5 minutes.

4. Per well of a 6 well plate, prepare “Tube B” containing 125 $\mu$L Opti-MEM^TM, 225 ng Recall plasmid (from Section 3.8), 275 ng dCas9-VPR plasmid and 2 $\mu$L$\mu$g DNA of p3000.

5. Slowly add “Tube B” dropwise to “Tube A” and carefully mix by gently inverting 10 times.

6. Incubate at room temperature for 20 minutes.

7. Add 225 $\mu$L of the transfection mix slowly and dropwise to each well of adherent cells.

8. 16-18 hours post-transfection, carefully remove media containing Lipofectamine^TM 3000/DNA complexes and slowly replenish with growth medium supplemented with 20% FBS without antibiotics.

9. 48-72 hours post-transfection, dissociate cells from the plate and wash cells with PBS twice at 300 x g for 5 minutes at 4 °C before resuspending in chilled FACS buffer.⁶⁴

10. Pass cells resuspended in FACS buffer through a 35 $\mu$m nylon mesh strainer into a 5 mL flow cytometry test tube and keep on ice.

11. Use control samples to set laser voltages on FSC-A, SSC-A, BFP, and GFP on FACS sorter such that nearly all cells are seen within FSC-A vs. SSC-A plot and both negative and positive populations can be seen and distinguished on the BFP and the GFP channel. Set compensations based on single positive populations.⁶⁵

12. Set sort gate on GFP and BFP double positive gate indicative of a recalled cell.⁶⁶

13. Sort cells in GFP and BFP double positive gate.⁶⁷

14. Maintain sorted cells in culture with complete growth medium.

10X Capture Variation #

3.1.1. Perform an extension reaction to generate double-stranded insert gRNA barcode DNA. Mix 10 $\mu$L NEB 5X Q5 Reaction Buffer, 1 $\mu$L of 10 mM dNTPs, 2 $\mu$L 100$\mu$M 10X-PrimeF-BgL-BbsI, 1 $\mu$L 100$\mu$M 10X-RevExt-BgL-BbsI and 0.5 $\mu$L to create a 50 $\mu$L reaction.

3.1.4. Digest 5-10 $\mu$g of 10X Capture vector backbone in a reaction containing 20 $\mu$L Digestion Buffer 3.1, 8 $\mu$L BbsI, and nuclease-fee water to 200 $\mu$L.

3.2.3. Prepare the PCR reaction to amplify barcodes and add Illumina indices by mixing 10 uL 5X Q5 Reaction Buffer, 1 $\mu$L 10 mM dNTPs, 2.5 $\mu$L PrimeF-PAS (from step 3.2.2), 2.5 $\mu$L PrimeR_10x (see Table 1), 0.5 $\mu$L Q5 polymerase, 50 ng plasmid DNA, and nuclease-free water to 50 $\mu$L.

3.6.1.3 Prepare the PCR reaction to amplify barcodes and add Illumina indices by mixing 10 uL 5X Q5 Reaction Buffer, 1 $\mu$L 10 mM dNTPs, 2.5 $\mu$L PrimeF-PAS (from step 3.2.2), 2.5 $\mu$L PrimeR_10x (see Table 1), 0.5 $\mu$L Q5 polymerase, up to 2 $\mu$g genomic DNA⁶⁸, and nuclease-free water to 50 $\mu$L.