1.1. Mouse Immunization
To obtain the B-cells that will undergo single-cell sequencing, mice will need to be immunized with the antigen of interest. We use BALB/c mice which has demonstrated faster spleen B-cell proliferation and a better overall humoral immune response than the commonly used C57BL/6 strain (13). However, any mouse strain used in immunological research should work with this method. Since this protocol is meant for a variety of possible antigens, the antibody titer of the mice has to be determined via indirect ELISA after the immunization (14). The day of peak antibody titer is determined by pooling the sera of 6 immunized mice after immunization. It is not absolutely necessary to determine the exact day of peak antibody titer for an individual mouse; we only want to approximate it so we can obtain a sufficient number of viable antigen-specific B cells for the 10x Genomics single-cell sequencing workflow (10,000 cells, 90% viability, 1,000 cells per µL). After the day of peak antibody titer is determined for your antigen of interest, it is recommended to immunize two separate mice for spleen extraction. A sufficient number of B-cells may be obtained with one spleen, but mouse spleens can vary quite significantly in size. The single cell suspension obtained from the spleens then undergoes RBC lysis and negative selection to remove cells other than B cells. Positive selection then has to be performed in order to isolate only the B cells that bind the antigen of interest. In this protocol, we use biotinylated BSA conjugated to the antigen and Streptavidin Nanobeads for positive selection. The isolated B cells are then sequenced through the 10x Genomics workflow in Section 3.2.
1.1.1 Mouse Immunization
1. Prepare 6 BALB/c mice to inject with the antigen of interest. Prepare 200 µL of vaccine for each injection, containing 3.3 nmol antigen solution, 20 µL of MPLA solution (1 mg MPLA/mL DMSO), and 172.4 µL PBS. Inject the vaccine subcutaneously under the scruff of the mice. The day before inoculating the mice, draw approximately 40 µL of blood from each of the mice and pool the sera to run an indirect ELISA (as a starting point for the antibody titer). (see Note [4]).
2. Perform a blood draw the next week, and then every other week afterwards (i.e., perform blood draws on days 7, 21, 35, 49, etc.). Perform two booster vaccinations on all of the mice on the weeks after the second and third blood draws (i.e., on days 14 and 28).
3. To run the indirect ELISA, apply the following procedure adopted from Wu et al, 2021 (13): Add 10 µg/mL of BSA-antigen conjugate in 0.05 M, pH 9.6 NaHCO3/Na2CO3 buffer containing 0.02% NaN3 to the 96-well plates. Incubate these plates overnight at 4 ºC. Wash these plates with PBS/0.5% Tween-20, and then add 100 µL of 1% BSA and PBS to each well at 22 ºC. Wait 1 hour for the block to occur. Wash the plates with PBS/0.5% Tween-20 and then incubate them with mouse sera dilutions in 0.1% BSA/PBS (add 100 µL per well of this diluted sera and use three wells per dilution). Incubate the plates at 37 ºC. Let the plates incubate for two hours and then wash them with PBS/0.5% Tween-20. Add a 1:2,000 dilution of the HRP-conjugated goat anti-mouse IgG in 100 µL of 0.1% BSA/PBS to the wells and incubate the plates for one hour at 37 ºC. Wash the plates with PBS/0.5% Tween-20. To each of the plates, add the following solution: 5 mg of TMB in 2 mL of DMSO plus 18 mL of citric acid buffer containing 20 µL of H2O2). After about fifteen minutes of letting the color form, add 50 µL H2SO4 (0.5 M) to quench. Measure the readout at 450 nm. Use regression analysis to determine the titer (see Note [5]). Perform all samples in triplicate.
4. Plot the antibody titer from the six mice over time to determine the day when levels reach their peak.
1.1.2 Harvesting B-cells from Immune-adapted Mice
5. Once the day of peak antibody titer is determined, obtain two separate BALB/c mice and inoculate them as described above, administering booster injections on days 14 and 28 after the initial inoculation. There is no need to perform blood draws for ELISA this time since the day of peak antibody titer is known. Euthanize the mice with CO2 on the day determined to have peak antibody titer (see Note [6]).
6. Sterilize the euthanized mice with 70% ethanol. Create incisions on the left ventral sides of the mice (approximately 2.5 cm in length) between the last rib and the hip joint using sterilized surgical scissors. Cut the skin, but do not cut the peritoneal wall yet.
7. Obtain two sterile petri dishes and fill them each with a 10 mL solution of RPMI 1640 culture medium, 10% Fetal Bovine Serum (FBS), 10 mM HEPES, 50 µM mercaptoethanol, 2 mM L-glutamine, 100 µg/mL penicillin G, and 1% penicillin-streptomycin.
8. Using a second sterilized pair of surgical scissors, create 2.5 cm long incisions in the exposed peritoneal walls with the same orientations as the previous skin incisions. Use sterilized forceps to pull the spleens through these incisions. Cleanly remove the connective tissue from the spleen using forceps or scissors and place the spleens in the prepared petri dishes.
9. Using the top side of a 5 mL syringe plunger, grind the spleens gently until they become a homogeneous slurry (see Note [7]).
10. Pipette the medium along with the spleen contents from the petri dishes to a single 50 mL centrifuge tube. Add 2 mL of the RPMI 1640 culture medium back into each of the petri dishes to rinse them, and then add the rinse to the centrifuge tube.
11. Centrifuge the contents of the 50 mL tube containing the spleens for 5 minutes at 1600 RPM and 4 ºC.
12. Aspirate the supernatant from the tube and then loosen the cell pellets by gently flicking the tube. Add 5 mL of red blood cell lysis buffer to the centrifuge tube. Incubate it on ice while gently shaking for 5 minutes. Dilute the contents of the centrifuge tube with PBS until the 50 mL mark is reached. Centrifuge for 5 minutes at 1600 RPM and 4 ºC.
13. Aspirate the supernatant again and loosen the cell pellets as mentioned in step 12. Add 20 mL of the RPMI 1640 culture medium to resuspend the cell pellets.
14. Acquire another 50 mL centrifuge tube and a 70 µm nylon mesh. Filter the contents of the original tube containing the cell pellets by pouring it into the new tube through the nylon mesh.
15. Count the cells with a hemocytometer (or cell counter) and use 0.4% trypan blue solution to check cell viability. Ensure that the cell viability is above 90%. You will need to immunize another pair of mice and extract their spleens on the day of peak antibody titer if the cell viability is less than 90% (i.e., restart with step 5).
1.1.3 Negative Selection for B-cell Isolation
16. Centrifuge the cell suspension for 5 minutes at 1,600 RPM and 4 ºC. Resuspend in an appropriate volume of MojoSortTM buffer. Adjust the cell concentration to 1 x 108 cells per mL using the buffer.
17. Perform a slightly modified version of the MojoSortTM protocol for negative selection, starting with the fourth step in the MojoSort TM protocol, but begin by aliquoting 300 µL of cell suspension instead of the 100 µL specified (15). This is to ensure a sufficient number of cells for the sequencing later in section 3.2. Aliquot the cell suspension into a new 14 mL polypropylene tube (do not use a standard centrifuge tube). Instead of adding 10 µL of the Biotin-Antibody cocktail and 10 µL of BioLegendTM Streptavidin Nanobead solution, as specified in the protocol, add 30 µL of each. DO NOT DISCARD THE SUPERNATANT—these are your cells of interest. Conduct everything else as specified in the protocol. Negative selection removes most cells which are not B cells.
18. Perform a cell count and viability check on the supernatant cells obtained from the negative selection using a hemocytometer and 0.4% trypan blue. Ensure well over 10,000 cells are present with a viability of over 90%. If fewer than 10,000 cells are present, repeat step 17 by aliquoting another 300 µL of cell suspension. If the viability is less than 90%, another pair of mice need to be immunized and have their spleens extracted on the day of peak antibody titer (see step 5).
1.1.4 Positive Selection for B-cell Isolation
19. Perform a modified version of the available MojoSortTM positive selection protocol beginning with its fourth step, but do not aliquot 100 µL of cell suspension as mentioned. Simply use the supernatant from the negative selection (16). Also, do not use the Biotin-conjugated antibody supplied in the MojoSortTM kit, since your desired cells are only the B-cells that bind your antigen of interest. Instead, use your antigen of interest conjugated to biotinylated BSA. The volume and concentration of biotinylated BSA antigen solution should be determined through titration for optimal results. Add 10 µL of BioLegendTM Streptavidin Nanobead solution for every 107 cells. Follow everything else as listed in the protocol.
20. Ensure the buffer of the cells contains no calcium or magnesium. This is needed for the 10x Genomics single cell sequencing. The MojoSortTM buffer should not contain calcium or magnesium, but if you are using a buffer with those ingredients, resuspend your cells in a buffer lacking them, such as a PBS + BSA buffer.
21. Perform a cell count and viability check using a hemocytometer and 0.4% trypan blue. Ensure you have at least 10,000 cells, with a concentration of 1,000 cells per microliter and a viability of above 90%. If you meet the viability but not the cell count criterion, start with step 17 by aliquoting another 300 µL of the cell suspension obtained prior to negative selection. If the viability criterion is not met, start by immunizing another pair of mice (see step 5).
22. Add an appropriate amount of DMSO (10%) to your cells as an anti-freeze agent, and package them in dry ice for shipping and storage prior to single cell sequencing.
1.2. Single-Cell Sequencing of BCRs
Single-Cell libraries for sequencing were generated using 10x Genomics’ Chromium Single Cell Immune Profiling platform (see Note [8] for alternatives). Sequencing was achieved using Illumina NovaSeq 6000 SP platform (17–19). Unless otherwise stated, the manufacturer’s recommended protocols throughout this section were implemented. First, gel beads-in-emulsion (GEMs) are generated to separate the collection of thousands of cells into pairs of single cells with unique barcoded gel beads suspended in partitioning oil. The vast majority of gel beads are unpaired to ensure only a single cell is paired to each gel bead barcode. Following the generation of GEMs, reverse transcription (RT) is performed to form cDNA from the lysed cells. The RT primer consists of Illumina’s read 1 (R1) sequencing primer, a cell-specific 10x barcode sequence, a transcript-specific unique molecular identifier (UMI), and a common template switch oligo (TSO) (see Figure 1B). Reagents and primers from RT are removed from cDNA generated in this step using silane magnetic beads. Using mouse B-cell specific primers that bind to the constant regions of the BCR, the cDNA libraries can be enriched. V(D)J sequencing libraries for BCRs were generated to include Illumina’s read 2 primer sequences through adaptor ligation after performing end repair and A-tailing on the enriched cDNA samples. Furthermore, using primers P5 and P7, the sampling indexes i5 and i7 were added to each molecule. Samples were cleaned-up using SPRIselect reagent kit. Gene Expression (GEX) sequencing libraries are then generated in a similar manner to the previous section. The resulting libraries must then be pooled for sample processing using Illumina’s NovaSeq platform, following manufacturer’s protocol for the XP workflow.
1.2.1 Prepare GEM Reaction Mix
1. Prepare the following RT master mix according to the number of samples being prepared (see Note [9]): 18.8 µL RT Reagent B, 7.3 µL Poly-dT RT Primer, 1.9 µL Reducing Agent B, 8.3 µL RT Enzyme C.
2. Transfer 36.3 µL of Master Mix into each tube of a PCR 8-tube strip on ice or cooling tube rack.
3. Assemble the Chromium Next GEM Chip K according to manufacturer instructions.
4. Based on the cell count and cell stock concentrations, transfer the appropriate volume of cell suspension and nuclease-free water to the Master Mix (see Note[10]). Each tube should have 75 µL in total. Pipette to mix.
1.2.2 Load the Chromium Next GEM Chip K
5. For each unused well, add 50% glycerol solution. 45 µL for row 3, 50 µL for row 2, and 70 µL for row 1.
6. Pipette 70 µL of Master Mix with Cells to each well in row 1. Ensure that no bubbles are introduced.
7. Prepare the gel beads by assembling the 10x Vortex Adapter to the tube strip holder with the gel bead Strip. Vortex for 30 seconds, then centrifuge the gel bead strip for 5 seconds. Ensure there are no bubbles in the tubes and secure the gel bead strip back in the holder with the lid.
8. After puncturing the foil seal of the gel bead tube, load Row 2 with 50 µL of gel beads. Wait 30 seconds.
9. Add 45 µL of partitioning Oil into row 3.
10. Attach gasket and add chip K to Chromium Controller or Chromium X/iX. Run the samples. While samples are being processed, place a PCR tube strip on ice.
11. After the approximately 18-minute cycle, remove the chip and discard the gasket. Ensure the volumes in rows 1 and 2 are equal to check if there are any clogs. During the cycle, place a PCR 8-tube strip on ice.
12. SLOWLY transfer 100 µL of GEMS from row 3 to the PCR tube strip. The liquid should appear as a uniform, opaque solution across all channels.
13. Transfer the tube strip to a thermal cycle, and run the samples according to the protocol in Table 1 of the Supplement.
1.2.3 Post GEM-RT Cleanup using Dynabeads
14. Add 125 µL of the recovery agent (dyed pink) to each sample at room temperature. Allow the mixture to separate into two phases for 2 minutes (see Note [11])
15. Remove 125 µL of the recovery agent/oil phase (dyed pink) from the bottom of the tube WITHOUT aspirating the sample within the clear aqueous phase (see Note [12]).
16. Prepare the following Dynabeads Cleanup Mix according to the following mixture, adjusting the total volume to the number of samples being prepared: 5 µL Nuclease-Free Water, 182 µL cleanup buffer, 8 µL Dynabeads MyOne SILANE, 5 µL Reducing Agent B.
a. Before adding the SILANE beads, vortex thoroughly for at least 30 seconds. If clumps remain, pipette to mix suspension.
17. Vortex Dynabeads Cleanup mix and add 200 µL of the mix to each sample. Pipette to mix. Incubate for 10 minutes at room temperature (with caps open).
18. While samples incubate, prepare Elution Solution 1, adjusting the total volume to the number of samples being prepared: 98 µL Buffer EB, 1 µL 10% Tween 20, 1 µL Reducing Agent B. Vortex to mix.
19. After samples incubate for 10 minutes, place tube strip/tubes on the 10x Magnetic Separator set to High until the solution is clear. (A white boundary layer between the two phases may form.) Remove the supernatant.
20. With the tubes on the magnetic separator, add 300 µL 80% ethanol to each. Wait for 30 seconds, then remove ethanol.
21. Centrifuge before placing the sample on the 10x Magnetic Separator Low position with the magnet set to the low setting.
22. Remove any remaining ethanol and allow the pellet to air dry for at least 2 minutes.
23. Immediately add 35.5 µL of Elution Solution 1 (from step 18) to the sample after removing the tube from the magnet. Pipette to mix until beads are fully resuspended (without introducing bubbles).
24. After incubating the sample for 1 minute at room temperature, place the samples on the magnetic separator set to low.
25. Once the solution clears, transfer 35 µL of each sample to a new 8-tube strip.
1.2.4 cDNA Amplification
26. Prepare the cDNA amplification mix, adjusting the total volume to the number of samples being prepared: 50 µL Amp Mix, 15 µL cDNA primers(see Note [13]).
27. Add 65 µL of the cDNA amplification mix to each 35 µL sample from step 25.
28. Pipette to mix, centrifuge briefly, and incubate samples according to the thermal cycler protocol in Table 2 of the Supplement.
a. The number of amplification cycles should be optimized based on the targeted cell recovery and the
cell type. Suggested initial guesses for the number of cycles for a given targeted cell recovery and cell
type are provided in Table 3 of the Supplement.
29. Store sample (see Note [14]), or proceed to the following step.
1.2.5 SPRIselect cDNA Cleanup
30. Add 60 µL of SPRIselect reagent (0.6X) to each sample and pipette to mix.
31. After incubating for 5 minutes at room temperature, place the tube strips on the magnetic separator with the magnet set to high. Once the solution is clear, remove the supernatant.
32. Add 200 µL of 80% ethanol to the pellet, wait 30 seconds, and then remove the ethanol. Repeat this wash step.
33. Centrifuge the tube strip and place on the magnetic separator with the magnet set to low.
34. Remove any residual ethanol and air dry for at least 2 minutes.
35. After removing the tubes from the magnet, add 45.5 µL Buffer EB. Pipette to mix.
36. After incubating for two minutes at room temperature, place the tube strip on the magnetic separator with the magnet set to high.
37. Once the solution clears, transfer 45 µL of the sample to the new 8-tube strip.
38. Store the sample (see Note [15]), or proceed to the following step.
1.2.6 cDNA QC and Quantification
39. Analyze the samples by running 1 µL of the undiluted sample on a Bioanalyzer (see Note[16])
a. For cells with low RNA content run 1 µL. But for high RNA content cells, use 1 µL of a 1:10 dilution in
ultra-pure water.
b. Quantify the cDNA yield for each sample.
1.2.7 V(D)J Amplification 1
40. Place cDNA product on ice.
41. Transfer 2 µL of each sample to a tube strip.
42. Prepare the following V(D)J Amplification 1 Reaction mix on ice, adjusting the total volume to the number of samples being prepared: 50 µL Amp mix, 48 µL B Cell Mix 1 v2.
43. Add 98 µL of the V(D)J Amplification 1 Reaction mix to each 2 µL sample. Pipette to mix and centrifuge briefly.
44. Incubate samples with the thermal cycle detailed in Table 4 of the Supplement.
45. Store samples at 4 ºC for up to 72 hours, or proceed to the following step.
1.2.8 SPRIselect post-V(D)J Amplification 1 Cleanup
46. Add 50 µL SPRIselect reagent (0.5X) to each sample. Pipette to mix.
47. After incubating samples at room temperature for five minutes, place the tube strip on the magnetic separator set to high.
48. Once the solution clears, transfer 145 µL of the supernatant to a new tube strip. DO NOT DISCARD SUPERNATANT.
49. Add 30 µL SPRIselect reagent (0.8X) to each sample. Pipette to mix.
50. After incubating for 5 minutes at room temperature, place the tube strip on the magnetic separator set to high.
51. Once the solution is clear, remove 170 µL supernatant, making sure not to aspirate any of the beads.
52. Add 200 µL 80% ethanol, wait 30 seconds, aspirate and discard the ethanol. Repeat this ethanol washing step two more times.
53. Centrifuge samples, then place sample on the magnetic separator set to low.
54. Remove any residual ethanol and add 35.5 µL buffer EB. Pipette to mix.
55. After incubating for 2 minutes at room temperature, place the tube strip on the magnetic separator set at low.
56. Once the solution supernatant is clear, transfer 35 µL of the sample to a new tube strip.
57. Store sample (see Note[17]) or proceed to the next step.
1.2.9 V(D)J Amplification 2
58. Prepare V(D)J Amplification 2 Reaction mix on ice, adjusting the total volume to the number of samples being prepared: 50 µL Amp Mix, 15 µL B Cell Mix 2 v2.
59. Add 65 µL of V(D)J Amplification 2 Reaction Mix to each sample. Pipette to mix and centrifuge briefly.
60. Run the samples with the thermal cycle detailed in Table 5 of the Supplement.
61. Store sample at 4 ºC for up to 72 hours, or proceed to the next step.
1.2.10 SPRIselect post-V(D)J Amplification 2 Cleanup
62. Add 50 µL SPRIselect reagent (0.5X) to each sample. Pipette to mix.
63. After incubating samples at room temperature for five minutes, place the tube strip on the magnetic separator set to high.
64. Once the solution is clear, transfer 145 µL of the supernatant to a new tube strip. DO NOT DISCARD SUPERNATANT.
65. Add 30 µL SPRIselect reagent (0.8X) to each sample. Pipette to mix.
66. After incubating for 5 minutes at room temperature, place the tube strip on the magnetic separator set to high.
67. Once the solution is clear, remove 170 µL supernatant, making sure not to aspirate any of the beads.
68. Add 200 µL 80% ethanol, wait 30 seconds, and remove the ethanol. Repeat this ethanol washing step two more times.
69. Centrifuge samples, then place samples on the magnetic separator set to low.
70. Remove any residual ethanol, and add 45.5 µL buffer EB. Pipette to mix.
71. After incubating for 2 minutes at room temperature, place the tube strip on the magnetic separator set at low.
72. Once the solution is clear, transfer 45 µL of the sample to a new tube strip.
73. Store sample (see Note [18]) or proceed to the next step.
74. Perform QC on the V(D)J Amplification with an Agilent Bioanalyzer High Sensitivity chip (see Note [19]).
a. Run 1 µL of each sample at a 1:5 dilution using ultra-pure water. If using RNA-rich cells, dilute the
samples further.
b. Determine the yield for each sample.
1.2.11 Fragmentation, End Repair, and A-tailing
75. Before starting step 76, pre-chill a thermal cycler to 4 ºC.
76. Pipette 50 ng of each sample into a tube strip set on ice.
a. Adjust volume to 20 µL with nuclease-free water if volume of 50 ng is less than 20 µL.
b. If 50 ng of the sample exceeds 20 µL, only use 20 uL of the sample.
77. Prepare the fragmentation mix, adjusting to the number of samples being prepared: 15 µL nuclease-free water, 5 µL fragmentation buffer, and 10 µL fragmentation enzyme. Thoroughly vortex the fragmentation buffer before adding.
78. Add 30 µL of the Fragmentation Mix into each tube containing 20 µL of sample. Pipette to mix and centrifuge briefly.
79. Transfer the samples into the thermal cycler and run the protocol detailed in Table 6 of the Supplement.
1.2.12 Adaptor Ligation
80. While the samples undergo fragmentation, prepare the following Adaptor Ligation Mix, adjusting to the number of samples being prepared: 20 µL ligation buffer, 10 µL DNA ligament, and 20 µL Adaptor Oligos.
81. After the protocol in step 79 is finished, add 50 µL of the Adaptor ligation mix to each of the samples. Pipette to mix and centrifuge briefly.
82. Run the samples with the thermal cycler protocol detailed in Table 7 of the Supplement.
1.2.13 SPRIselect post-Ligation Cleanup
83. Add 80 µL of 0.8X SPRIselect reagent to each sample. Pipette to mix.
84. After incubating for 5 minutes at room temperature, place the tube strip on the magnetic separator set on high. Remove the supernatant when the solution is clear.
85. Add 200 µL 80% ethanol to the pellet, wait 30 seconds, and remove the ethanol. Repeat this ethanol washing step.
86. Centrifuge the tube strips, and place the tubes on the magnetic separator set to low. Remove any residual ethanol, and air dry for 2 minutes.
87. Add 30.5 µL Buffer EB. Pipette to mix until beads are thoroughly resuspended.
88. After incubating for 2 minutes at room temperature, place the tube strips on the magnetic separator set to low.
89. Once the solution is clear, transfer 30 µL of the sample to a new tube strip.
1.2.14 Sample Index PCR
90. Add 50 µL of the Amp Mix to 30 µL sample.
91. Add 20 µL of an individual Dual Index TT Set A to each well and record the well ID used. Ensure that the well IDs are not repeated in a single run. Pipette to mix and centrifuge briefly.
92. Incubate the samples with the thermal cycler protocol detailed in Table 8 of the Supplement.
93. Store samples at 4 ºC for up to 72 hours or proceed to the following step.
1.2.15 SPRIselect post-Sample Index PCR Cleanup
94. Add 80 µL of 0.8X SPRIselect reagent to each sample. Pipette to mix.
95. After incubating for 5 minutes at room temperature, place the tube strip on the magnetic separator set on high. Remove the supernatant.
96. Once the solution is clear, add 200 µL 80% ethanol to the pellet, wait 30 seconds, and remove the ethanol. Repeat this ethanol washing step two more times.
97. Centrifuge the tube strips and place the tubes on the magnetic separator set to low.
98. Remove any residual ethanol, and air dry for 2 minutes.
99. Add 35.5 µL Buffer EB. Pipette to mix until beads are thoroughly resuspended.
100. After incubating for 2 minutes at room temperature, place the tube strips on the magnetic separator set to low.
101. Once the solution is clear, transfer 35 µL of the sample to a new tube strip.
102. Store sample at 4 ºC for up to 72 hours or at -20 ºC for long-term storage.
103. Analyze 1 µL sample diluted by 1:10 on an Agilent Bioanalyzer High Sensitivity chip (see Note [20])
a. Determine the average fragment size from the trace for library quantification.
1.2.16 GEX Library Fragmentation, End Repair, and A-tailing
104. Before starting step 108, pre-cool the thermal cycler block to 4 ºC.
105. Add 50 ng of the GEX sample into a tube within a tube strip that has been chilled on ice.
a. If the total volume is less than 20 µL, add enough nuclease-free water for a total volume of 20 µL.
b. If the volume of 50 ng is greater than 20 µL, only add 20 µL of the sample into the tube.
106. Vortex the Fragmentation Buffer before preparing the following Fragmentation Mix, adjusting the total volume to the number of samples being prepared: 15 µL nuclease-free water, 5 µL Fragmentation Buffer, 10 µL Fragmentation Enzyme.
107. Transfer 30 µL of Fragmentation Mix into each of the 20 µL samples.
108. Run the samples with the thermal cycler protocol detailed in Table 9 of the Supplement.
1.2.17 SPRIselect post-Fragmentation, End Repair, and A-tailing Cleanup
109. After the protocol is complete, add 30 µL 0.6X SPRIselect Reagent to each sample. Pipette to mix.
110. After incubating the SPRIselect beads with the samples for 5 minutes at room temperature, place the tube strip on the magnetic separator set to high. DO NOT DISCARD THE SUPERNATANT.
111. Once the solution is clear, transfer 75 µL of the supernatant to a new tube strip.
112. Add 10 µL of 0.8X SPRIselect reagent to each sample. Pipette to mix.
113. After incubating for 5 minutes at room temperature, place the tube strip on the magnetic separator set to high.
114. Once the solution is clear, remove 80 µL of the supernatant. DO NOT DISCARD ANY OF THE BEADS.
115. With the tube strip on the magnetic separator, add 125 uL 80% ethanol to the pellet and remove the ethanol after 30 seconds. Repeat this washing step one more time.
116. Remove the ethanol and then remove the tube strip from the separator.
117. Add 50.5 µL Buffer EB. Pipette to mix. Incubate for 2 minutes at room temperature.
118. Place the tube strip on the magnetic separator set to high. Once the solution is clear, transfer 50 µL of each sample to a new tube strip.
1.2.18 GEX Adaptor Ligation
119. Add 50 µL of the following Adaptor Ligation Mix to each sample, pipetting to mix: 20 µL Ligation Buffer, 10 µL DNA Ligase, 20 µL Adaptor Oligos.
120. Incubate samples with the thermal cycler protocol detailed in Table 10 of the Supplement.
1.2.19 SPRIselect – post-Ligation Cleanup
121. Add 80 µL 0.8X SPRIselect Reagent to each sample, pipette to mix.
122. After incubating the samples for 5 minutes at room temperature, place the tube strip on the magnetic separator set to high. Once the solution is clear, remove the supernatant.
123. Add 200 µL 80% ethanol to the pellet, wait 30 seconds, and remove the ethanol. Repeat this washing step one more time.
124. Centrifuge briefly and place the tube strip on the magnetic separator set to low.
125. Remove any remaining ethanol and air dry for 2 minutes.
126. Remove the tubes from the magnet, and add 30.5 µL Buffer EB, pipetting to mix.
127. Incubate the samples for 2 minutes at room temperature, and place the tubes on the magnetic separator set to low.
128. Once the solution is clear, transfer 30 µL of each sample to a new tube strip.
1.2.20 GEX Sample Index PCR
129. Add 50 µL Amp mix to each sample, and add 20 µL of Dual Index TT Set A to each well. Pipette to mix.
130. Incubate the samples with the thermal cycler protocol detailed in Table 11 of the Supplement. See Table 12 for the number of denaturing, annealing, or extension cycles based on cDNA input.
131. Store sample at 4 ºC for up to 72 hours, or proceed to the next step.
1.2.21 SPRIselect post-GEX Sample Index PCR Cleanup
132. Add 60 µL 0.6X SPRIselect reagent to each sample. Pipette to mix.
133. After incubating for 5 minutes at room temperature, place the tube strip on the magnetic separator set to high. DO NOT DISCARD THE SUPERNATANT.
134. Once the solution is clear, transfer 150 µL of the supernatant to a new tube strip.
135. Add 20 µL 0.8X SPRIselect reagent to each sample. Pipette to mix.
136. After incubating for 5 minutes at room temperature, place the tubes on the magnetic separator.
137. Once the solution is clear, remove 165 µL of the supernatant, ensure that none of the beads are aspirated.
138. With the tubes still on the magnet, wash the pellet with 200 µL 80% ethanol. Wait 30 seconds before removing the ethanol. Repeat this wash step one more time.
139. Centrifuge samples briefly before placing tubes back on the magnetic separator set to low.
140. Remove the remaining ethanol before adding 35.5 µL Buffer EB.
141. Remove the tube strip on the magnet, and pipette to mix.
142. After incubating for 2 minutes at room temperature, return the tubes to the magnetic separator set to low.
143. Once the solution is clear, transfer 35 µL of each sample to a new tube strip.
144. Store at 4 ºC for up to 72 hours or -20 ºC for long-term storage.
145. Analyze 1 µL of the sample diluted 1:10 on an Agilent Bioanalyzer High Sensitivity chip (see Note [21]).
146. The V(D)J library and GEX library are subsequently pooled in a 1:7 ratio using a single SP 2x150 bp paired end lane from Illumina’s NovaSeq 6000 SP platform using the XP workflow (see Note [22]).
1.3. Lead Clone Characterization and Refinement
To limit the burden of experimentally characterizing each potential clone, this section entails an overview of how the raw sequencing data produced by Illumina sequencing can be interpreted as a list of clonotypes. After converting the BCL file into the FastQ file format, 10x Genomics’ cellranger program can be used to generate sequences and counts of paired clonotypes from V(D)J libraries after performing alignment, filtering, and counting operations (17, 19). While not extensively detailed in this Method, several computational tools and workflows can be implemented at this stage to reduce the number of clones to characterize experimentally based on various developability criteria. In the notes section below, we provide some suggestions to incorporate tools to consider humanness (see Note [23]), binding affinity (see Note [24]), and developability broadly (see Note [25]).
1. The FastQ sequencing file can be generated from the raw data produced in the sequencing workflow using bcl2fastq2 with the PE150 format. After installing bcl2fastq2 into a compatible Linux/CentOS workstation, the following command can be run with the necessary input base call files (.cbcl), filter files (.filter), cluster location files (s.locs), and run info file defined with the --input-dir option (see Note [26]):
nohup /path/to/bcl2fastq --runfolder-dir /path/to/run_folder \
--output-dir /path/to/output_dir \
--input-dir /path/to/input_directory
2. Prepare the following lines in a CSV file to use as an input for the cellranger multi pipeline (see Note[27]):
[gene-expression]
reference, /path/to/gex_data
[vdj]
reference, /path/to/vdj_data
[libraries]
fastq_id, fastqs, lanes, feature_types, subsample_rate
GEX_fastqs_id, /path/to/GEX_fastq, 1|2, Gene Expression,
VDJ_B_fastqs_id, /path/to/vdj_fastqs, 1|2, VDJ-B,
3. Run the cellranger multi pipeline (for documentation, see Note[28]):
cellranger multi --id=[run ID string (see Note [29])] \
--csv=/path/to/csv_input_file
4. Much of the 10x Genomics sequencing data obtained from the cellranger multi pipeline can be downloaded in CSV format, and the amino acid or DNA sequences of the framework regions (FWRs) and complimentary determining regions (CDRs) can be extracted from the output file “consensus_annotations.csv” (see Note[30]). Once these sequences are extracted, they can be concatenated in their numbered order (i.e., fwr1, cdr1, fwr2, cdr2, fwr3, cdr3, fwr4) to form VH and VL chains. These chains can be paired with their respective clonotype ID (e.g., clonotype1, clonotype2, etc.), the chain ID (e.g., IGK, IGH, IGL) and the number of reads for each chain. These data should be exported to a separate CSV file containing the clonotype ID in the first column, the concatenated VH or VL sequence in the second, the reads per each chain in the third, and the chain ID in the fourth. Once this CSV file is ready, you can perform the steps that follow to generate a chimeric single chain Fab provided you also have the sequence of a human IgG1 scFab that you can obtain CH, CL, and linker sequences from.
5. Run the available SequenceBuilder MATLAB script on the CSV file made in step 4, making sure to include the correct name of the CSV file in the script (the default name is 10XGEN.xlsx) (see Note[31]). Run the script to generate the corresponding VH and VL chains for each clonotype in separate CSV outputs. This script will go through the data for each of the clonotypes, and if any of them happen to have multiple light chains listed, it will select the one with the most reads.
6. Obtain the amino acid or DNA sequences of the constant heavy (CH), constant light (CL) and linker regions of the human IgG1 scFab. Make sure the antibody numbering system used in the 10x Genomics sequencing data is consistent with the numbering system used by any software that identifies the CH, CL and linker regions of the IgG1 Fab.
7. Assemble all of the obtained amino acid or DNA sequences in the following order to obtain the amino acid or DNA sequences of the chimeric scFabs: VL, CL, linker, VH, CH. Three-dimensional structures of the scFabs can now be obtained using homology modeling software. These chimeric scFabs can offer a starting point for further computational refinement and development of expressed antibodies with increased humanness and reduced immunogenicity.
8. For generating scFab sequences with greater humanness, you can choose to extract only the CDR regions from the consensus_annotations.csv file. These CDR regions will have to be concatenated with the framework regions of the human IgG1 scFab (in the same order as presented in step 4) so that VH and VL chains are obtained. Make a separate CSV with these VH or VL sequences, chain IDs, clonotype IDs, and reads as mentioned in step 4. Make sure the numbering systems for the 10X Genomics data and the IgG1 Fab are the same. Run the MATLAB script and then follow steps 6 and 7 as mentioned above.
1.4. Library Generation and YSD Expression
After determining which antibodies to experimentally characterize, the VH and VL domains created in part 3.3 can be isolated to be cloned into yeast display vectors. The VH and VL inserts can be either directly amplified from cDNA libraries generated in section 3.2 or purchased as synthetic DNA fragments. A standard T4 ligation-based strategy is presented below, but other cloning methods may be substituted. After propagating plasmid in competent E. coli (NEB5α), the expression vector is transformed into yeast via electroporation or EZYeast transformation (see Note[32]).
1.4.1 Prepare VH and VL Inserts
1. VH and VL domains can be prepared by direct PCR amplification from cDNA libraries or ordered as synthetic DNA fragments (see Note [33]). In either case, the insert should be flanked with NheI and BamHI for downstream steps.
2. Prepare the inserts by incubating the restriction digest mix detailed in Table 13 of the Supplement overnight at 37 ºC.
3. Isolate the desired fragment with a DNA cleanup kit or gel extraction kit (after performing gel electrophoresis on RE digest products) according to the manufacturer’s instructions.
4. Quantify insert concentration.
1.4.2 Prepare Yeast Expression Vector
5. Pick a single colony of pCT-CON2 expression vector and inoculate 7 mL of LB-amp overnight (see Note [34]).
6. Isolate plasmid from the overnight culture with a plasmid preparation kit of your choice, following the manufacturer’s instructions.
7. Quantify plasmid concentration and purity.
8. Prepare the restriction digest mix detailed in Table 14 of the Supplement to linearize the vector.
9. Incubate RE digest mix overnight at 37 ºC.
10. Isolate the desired linearized DNA with a DNA cleanup kit or gel extraction kit (after performing gel electrophoresis on RE digest products) according to the manufacturer’s instructions.
11. Quantify linearized DNA concentration and purity via A260:A280 ratio.
12. Store products at -20 ºC for long-term storage or proceed to the following step.
1.4.3 Ligation
13. Verify the concentration and purity of Insert and linearized backbone DNA.
14. Prepare the ligation reaction mix detailed in Table 15 of the Supplement.
15. Incubate at room temperature for 10 minutes.
1.4.4 Transformation
16. Transform 5 µL of ligation reaction mix product into 50 µL NEB5α competent cells, following the manufacturer’s recommended protocol (20). Store the remaining ligation product at -20 ºC after heat inactivating T4 ligase by incubating the reaction mix at 65 ºC for 10 minutes.
17. Thaw NEB5α cells on ice.
18. Pipette 5 µL of the ligation reaction mix to 50 µL of NEB5α competent cells.
19. Incubate the ligation mix-cell mixture on ice for 30 minutes.
20. Heat shock cell mixture at 42 ºC for exactly 30 seconds.
21. Place cells on ice for 5 minutes.
22. Add 950 µL SOB (alternatively SOC) media and incubate mixture at 37 ºC for 1-2 hours in a shaking incubator set to 250 rpm.
23. Plate 50-200 µL of cells on LB agar plate.
24. Incubate overnight at 37 ºC.
25. Verify clones by colony PCR and sanger sequencing.
1.4.5 Prepare Plasmid
26. Inoculate 7 mL of LB supplemented with ampicillin with a single colony from the LB-ampicillin agar plate from step 16.
27. Incubate overnight at 37 ºC.
28. Isolate desired plasmid with a plasmid prep kit of your choice, following manufacturer’s suggested protocol. Quantify plasmid yield and purity.
1.4.6 Transform Clones into Yeast: Option A - Electroporation
29. Grow an overnight colony of EBY100 yeast on YPD media at 30 ºC, shaking 250 rpm.
30. The following morning, for each sample, inoculate 10 x 107 cells into 100 mL of YPD.
31. Grow cells at 30 ºC, 250 rpm until a density of 1.3-1.5 X 107 cells/mL. (This step should take ~6-8 hours, assuming a doubling time of 1.5 hours). (see Note [35])
32. Pellet cells (2,250 rcf for 3 minutes) and discard the supernatant.
33. Wash cells with cold dH2O twice, pelleting cells at 2,250 rcf for 3 minutes to discard the supernatant.
34. Wash the cells once with 25 mL cold Buffer E.
35. Resuspend cells in 25 mL of a solution containing the following: 100 mM lithium acetate, 10mM Tris, pH 7.5, 1mM EDTA, 30% PEG 8000, 10 mM DTT (see Note [36])
36. Incubate cells for 30 minutes at 30 ºC, shaking at 250 rpm.
37. Centrifuge cells at 2,250 x g at 4 ºC for 3 minutes. Discard the supernatant.
38. Wash cells with 25 mL col buffer E. After centrifuging cells at 2,250 x g at 4 ºC for 3 minutes, discard supernatant.
39. Resuspend each sample in 1 mL of cold Buffer E, and transfer samples to 1.5 mL tubes.
40. Centrifuge at 5,000 x g at 4 ºC for 1 minute. Discard supernatant.
41. Wash cells twice with 1 mL cold Buffer E, centrifuging samples at 5,000 x g at 4 ºC for 1 minute.
42. After discarding the supernatant from the last wash, resuspend to cells in 300 µL of cold Buffer E.
43. Add 6 µg of your desired clone (or ~1.5 pmol) to the cells.
44. Transfer cell-DNA mixture to electroporation cuvette. Incubate on ice for 5 minutes.
45. Remove any condensation from the outside of the cuvette with a paper towel.
46. Pulse cuvette at 25 µF, 1.2 kV with a time constant of ~4-45 ms.
47. Immediately add 1 mL of room temperature YPD to cuvette, and chill cuvette on ice.
48. Transfer cell-DNA mixture to 15 mL conical tube with 4 mL YPD.
49. Incubate at 30 ºC for 1-2 hours in a shaking incubator set to 250 rpm.
50. Centrifuge cells at 1,300 x g for 1 minute.
51. Resuspend cells in 1 mL SD-/Trp and transfer cells to a baffled flask with 100 mL SD-/Trp.
52. Plate cells on SD/-Trp plates, creating serial dilutions ranging from 100x to 2,000x dilutions to determine transformation efficiency.
53. Incubate plates at 30 ºC. Cells in baffled flasks should be incubated for at least 16 hours at 30 ºC and 250 rpm.
1.4.7 Transform Clones into Yeast: Option B - EZYeast Transformation
54. Grow EBY100 cells in 10 mL YPD broth to an OD600 of 0.8-1.0 (mid-log phase).
55. Pellet cells at 500 x g for 4 minutes. Discard supernatant.
56. Wash cells with 10 mL EZ 1 solution, pellet cells at 500 x g for 4 minutes, and discard the supernatant.
57. Resuspend pellet with 1 mL EZ 2 solution.
58. Add 0.5-2 µg (less than 5 µL total volume) to 50 µL of competent cells with 500 µL EZ 3 solution. Mix thoroughly.
59. Incubate at 30 ºC for 45 minutes, vertexing 2-3 times to mix.
60. Pellet cells at 1,500 x g for 3 minutes. Discard supernatant.
61. Wash cells with 1 mL PBSA, pellet cells at 1,500 x g for 3 minutes, and discard the supernatant.
62. Resuspend cells in 3 mL of YPD media.
63. Incubate cells at 30 ºC for 1 hour.
64. Pellet cells at 1,500 x g for 3 minutes and remove supernatant.
65. Wash cells with 1 mL of PBSA, pellet cells at 1,500 x g for 3 minutes, and resuspend cells in 5 mL of SD.
66. Plate 50 µL of cells suspended in SD media to characterize transformation efficiency.