B-Cell Receptor Sequencing with UMI Protocol

RNA Extraction

1. Cell collection: Transfer cells to an appropriate tube, and pellet by centrifugation for 5 min at 300 ×g. Remove supernatant.
2. Homogenize and lyse sample by adding 350 μL buffer LBP to the cell pellet. Mixing the cells with a lysis buffer is usually sufficient for complete lysis.
3. Remove gDNA and filtrate lysate: Place the NucleoSpin® gDNA Removal Column (yellow ring) in a 2 mL collection tube, transfer the homogenized lysate to the NucleoSpin® gDNA Removal Column, and centrifuge for 30 s at 11,000 × g. Discard the column and continue with the flowthrough.
4. Adjust RNA binding conditions: Add 100 μL binding solution BS to the flowthrough, and mix well by moderate vortexing or by pipetting up and down several times. After addition of binding solution BS, a stringy precipitate may become visible which will not affect the RNA isolation. Be sure to disaggregate any precipitate by mixing and load all of the precipitate on the column as described in the following step. Do not centrifuge the lysate after addition of binding solution before loading it onto the column in order to avoid pelleting the precipitate.
5. Bind RNA: Transfer the whole lysate (~450 μL) to the NucleoSpin® RNA Plus Column (light blue ring) preassembled with a collection tube. Centrifuge for 15 s at 11,000 × g.
6. Wash and dry silica membrane:
(a) First wash: Add 200 μL buffer WB1 to the NucleoSpin® RNA Plus Column. Centrifuge for 15 s at 11,000 × g. Discard the flowthrough with the collection tube, and place the column into a new 2 mL collection tube.
(b) Second wash: Add 600 μL buffer WB2 to the NucleoSpin® RNA Plus Column. Centrifuge for 15 s at 11,000 × g. Discard flowthrough, and place the column back into the collection tube.
(c) Third wash: Add 250 μL buffer WB2 to the NucleoSpin® RNA Plus Column. Centrifuge for 2 min at 11,000 × g to dry the membrane completely. Place the column into a nuclease-free 1.5 mL collection tube.
7. Elute RNA: Add 30 μL RNase-free H2O and centrifuge at 11,000 × g for 1 min. Add an additional 30 μL RNase-free H2O to the column, and centrifuge again at 11,000 × g for 1 min.
8. After RNA extraction, if sample size is not limiting, we recommend evaluating total RNA quality using the Agilent RNA 6000 Pico Kit or an equivalent platform. Refer to the manufacturer for instructions.

First-Strand cDNA Synthesis

1. Thaw the First-Strand Buffer at room temperature. Thaw BCR enhancer, SMART UMI Oligo, and dT Primer on ice. Gently vortex each reagent to mix and centrifuge briefly. Store all but the First-Strand Buffer on ice. Remove the SMARTScribe reverse transcriptase and RNase inhibitor from the freezer immediately before use, centrifuge briefly, and store on ice.
2. Preheat the thermal cycler to 72 °C.
3. On ice, prepare samples and controls in nuclease-free thin-wall PCR tubes, plates, or strips by adding the reagents.
4. Mix by gently vortexing and then centrifuge briefly.
5. Incubate the tubes at 72 °C in the preheated, heated-lid thermal cycler for 3 min. During this incubation, prepare the RT Master Mix.
6. At room temperature, prepare RT Master Mix.
7. Mix the RT Master Mix well by gently pipetting up and down, and then centrifuge briefly.
8. Immediately after the 3-min incubation at 72 °C, place the samples on ice for 2 min.
9. Reduce the temperature of the thermal cycler to 42 °C.
10. Add 7.5 μL of the RT Master Mix to each reaction tube. Mix the contents of each tube by pipetting gently and centrifuge briefly.
11. Place the tubes in a thermal cycler with a heated lid, preheated to 42 °C. Run the following program: 42 °C, 90 min; 70 °C, 10 s; 4 °C hold.

First-Round Amplification

1. Thaw 5× PrimeSTAR GXL buffer, dNTP mix, primers, and nuclease-free water on ice. Gently vortex each reagent to mix and centrifuge briefly. Store on ice. Remove the PrimeSTAR GXL DNA polymerase from the freezer immediately before use, gently pipet to mix, centrifuge briefly, and store on ice.
2. Prepare a PCR1 Master Mix for each IgG/IgM/IgK/IgL chain of interest, by combining the following in the order shown, on ice. Gently vortex to mix and centrifuge briefly.
3. Add 46 μL of the appropriate IgG/IgM/IgK/IgL PCR1 Master Mix to nuclease-free, thin-wall 0.2-mL PCR plate/tube(s).
4. Add 4 μL of first-strand cDNA from Subheading 3.3 to the corresponding tube(s) containing PCR1 Master Mix. Gently vortex to mix, and centrifuge briefly.
5. Place the plate/tube(s) in a preheated thermal cycler with a heated lid, and run the following program (lid temperature: 105 °C): 95°C 1 min; 98°C 10 s, 60°C 15 s, and 68°C 45 s (18 cycles); 4°C hold.

Second-Round PCR Amplification

1. Thaw 5X PrimeSTAR GXL buffer, dNTP Mix, primers, and nuclease-free water on ice. Gently vortex each reagent to mix and centrifuge briefly. Store on ice. Remove the PrimeSTAR GXL DNA polymerase from the freezer immediately before use, gently pipet mix, centrifuge briefly, and store on ice.
2. For each IgG/IgM/IgK/IgL chain of interest, prepare a PCR2 Master Mix by combining the following in the order shown, on ice. Gently vortex to mix and centrifuge briefly.
3. For each reaction, add 48 μL of PCR2 Master Mix to nuclease-free, thin-wall, 0.2-mL PCR plate/tube(s).
4. Add 1 μL of appropriate PCR1 product to each corresponding PCR 2 tube.
5. Add 1 μL of the appropriate hBCR PCR2 Universal Forward 1–12 primer to each sample. Gently vortex to mix and centrifuge briefly.
6. Place the plate/tube(s) in a preheated thermal cycler with a heated lid, and run the following program (lid temperature: 105 °C): 95°C 1 min; 98°C 10 s, 60°C 15 s, 68°C 45 s (X* cycles); 4°C Hold.

Purification of Amplified Libraries

1. Vortex NucleoMag beads until evenly mixed, and then add 25 μL of the NucleoMag beads to each sample.
2. Mix thoroughly by gently pipetting the entire volume up and down at least ten times.
3. Incubate at room temperature for 8 min to let the DNA bind to the beads.
4. Briefly spin the samples to collect the liquid from the side of the tube or sample well. Place the samples on the magnetic separation device for ~5 min or longer until the liquid appears completely clear, and there are no beads left in the supernatant. The time required for the solution to clear will depend on the strength of the magnet.
5. While the reaction tubes are sitting on the magnetic separation device, use a pipette to transfer the supernatant (which contains your library) to clean PCR tubes.
6. Remove the tubes containing the beads from the magnetic separation device, and discard them.
7. Add 10 μL of NucleoMag beads to each tube containing supernatant.
8. Mix thoroughly by gently pipetting the entire volume up and down at least ten times.
9. Incubate at room temperature for 8 min to let the DNA bind to the beads.
10. Place the tubes on the magnetic separation device for ~10 min or until the solution is completely clear.
11. While the tubes are sitting on the magnetic separation device, remove the supernatant with a pipette and discard it.
12. Keep the tubes on the magnetic separation device. Add 200 μL of freshly made 80% ethanol to each sample, without disturbing the beads, to wash away contaminants. Wait for 30 s, and use a pipette to carefully remove the supernatant containing contaminants. The library will remain bound to the beads during the washing process.
13. Repeat the ethanol wash (step 12 of this section) once more.
14. Briefly spin the tubes (~2000 × g) to collect the remaining liquid at the bottom of each tube. Place the tubes on the magnetic separation device for 30 s, and then remove all remaining liquid with a pipette.
15. Let the sample tubes rest open on the magnetic separation device at room temperature for ~2–2.5 min until the pellet appears dry and is no longer shiny. You may see a tiny crack in the pellet.
16. Once the bead pellet has dried, remove the tubes from the magnetic separation device, and add 17 μL of elution buffer to cover the pellet. Mix thoroughly by pipetting up and down to ensure complete bead dispersion.
17. Incubate at room temperature for at least 5 min to rehydrate.
18. Briefly spin the samples to collect the liquid from the side of the tube or sample well. Place the samples back on the magnetic separation device for 2 min or longer until the solution is completely clear.
19. Transfer clear supernatant containing purified BCR /IG library from each tube to a nuclease-free, low-adhesion tube. Label each tube with sample information and store at −20 °C.

Sequencing

1. Dilute each library to 4 nM in nuclease-free water. To avoid pipetting errors, use at least 2 μL of each original library for dilution.
2. Pool the diluted libraries by combining an equal amount of each library in a low-bind 1.5-mL tube. Mix by vortexing at low speed or by pipetting up and down. Use at least 2 μL of each diluted library to avoid pipetting error.
3. Use a 5 μL aliquot of the 4-nM-concentration-pooled libraries. Follow the library denaturation protocol according to the latest edition of your Illumina sequencing instrument's user guide.

Reference:

1. Gupta N, Marquez S, Soto C, et al. Bulk Sequencing from mRNA with UMI for Evaluation of B-Cell Isotype and Clonal Evolution: A Method by the AIRR Community[M]//Immunogenetics. Humana, New York, NY, 2022: 345-377.