Superscript 4 enzyme

The antibody cloning of Env-sorted single B cells was conducted as follows. A mix containing 3 μl of random hexamers (GeneLink), 2 μl of deoxynucleoside triphosphates (dNTPs), and 1 μl of SuperScript IV enzyme (Thermo Fisher) was added to each well of a single-cell-sorted 96-well plate that underwent thermocycling according to the program outlined in the SuperScript IV protocol, resulting in 25 μl of cDNA for each single cell. cDNA (5 μl) was then added to a PCR mix containing 12.5 μl of 2× multiplex PCR mix (Qiagen), 9 μl of H₂O, 0.5 μl of forward primer mix, and 0.5 μl of reverse primer mix (mouse [105 (link)] and rabbit [55 (link)]) for HCs and KCs within each well. A second PCR was then performed using 5 μl of the first PCR as the template and respective primers (mouse [105 (link)] and rabbit [55 (link)]) utilizing the same recipe as the first PCR. The PCR products were run on a 1% agarose gel, and those with correct HC and KC bands were then used for Gibson ligation (New England Biolabs), cloning into IgG expression vectors, and transformation into competent cells. Mouse and rabbit MAbs were expressed by the transient transfection of ExpiCHO cells (Thermo Fisher) with equal amounst of paired HC and KC plasmids and purified from the culture supernatant after 12 to 14 days using protein A bead columns (Thermo Fisher).

Antibody variable gene mRNA transcripts (VH, Vk, Vλ) were amplified by RT-PCR as described previously (21). Briefly, cDNA was synthesized using SuperScript IV enzyme (ThermoFisher Scientific), followed by two rounds of nested PCRs. The second cycle of nested PCR added 40 base pairs of 5′ and 3′ homology to restriction enzyme-digested S. cerevisiae expression vectors to enable homologous recombination during transformation. PCR-amplified variable gene DNA was chemically transformed into competent yeast cells via the lithium acetate method and yeast were plated on selective amino acid drop-out agar plates (44). Transformed yeast colonies were picked for sequencing, recombinant antibody expression, and characterization.
For clonal lineage analysis, clonally expanded antibodies were defined by the following criteria: identical heavy and light chain germline genes, identical HCDR3 lengths, and >80% identical HCDR3 protein sequence.

Sequences from RBD-specific MBCs from healthy donors were in part obtained from our previous analysis of RBD-specific MBCs after mRNA vaccination (Sokal et al., 2021a (link)). All other sequences were obtained through single-cell sequencing. RBD-specific MBCs were single-cell sorted in 4 µl lysis buffer containing PBS (Gibco), dithiothreitol (Thermo Fisher Scientific), and RNAsin (Promega). A reverse transcription step was then performed using the SuperScript IV enzyme (Thermo Fisher Scientific) in 14 μl final volume (42°C 10 min, 25°C 10 min, 50°C 60 min, 94°C 5 min) with 4 µl of RNA in lysis buffer and random hexamers (Thermo Fisher Scientific). A PCR was further performed based on the protocol established by Tiller et al. (2008) (link). Briefly, 3.5 μl of cDNA was used as template and amplified in a total volume of 40 μl with a mix of forward L-VH primer (Table S1 B) and reverse Cγ primer by using the HotStar Taq DNA polymerase (Qiagen) and 50 cycles of PCR (94°C 30 s, 58°C 30 s, 72°C 60 s). Then a second 50-cycle PCR using 5′AgeI VH primer mix and CHG-D1 3′ primer was performed before sequencing (Table S1 D).
PCR products were sequenced with the reverse primer CHG-D1 and read on ABI PRISM 3130XL genetic analyzer (Applied Biosystems). Sequence quality was verified with the CodonCode Aligner software (CodonCode Corporation).

Human antibody variable gene transcripts (VH, Vκ, Vλ) were amplified by reverse transcription polymerase chain reaction (RT-PCR) using SuperScript IV enzyme (Thermo Scientific Cat# 18090050) followed by nested PCR using cocktails of variable region and IgM-, IgD-, IgA- and IgG-specific constant-region primers with HotStarTaq Plus DNA Polymerase (Qiagen Cat# 203646), as previously described (40 (link)). The primers used in the second round of nested PCR contained 40 base pairs of 5′ and 3′ homology for linearized yeast expression vectors to allow cloning by homologous recombination. Amplified variable gene transcripts were transformed into in S. cerevisiae using the lithium acetate method for chemical transformation (41 (link)). For each transformation reaction, 1x10⁶ yeast cells were mixed and incubated with 240 μl of polyethylene glycol (PEG) 3350 (50% w/v) (Sigma-Aldrich, Cat#202444), 36 μl of 1M lithium acetate (Sigma Aldrich, Cat#517992), 10 μl of denatured salmon sperm DNA (Invitrogen, Cat#15632011), 67 μl sterile water, 200 ng of the digested expression vectors and 10 μl each of unpurified VH and VL PCR products at 42°C for 45 min. Following transformation, the yeast were washed twice with sterile water, resuspended in selective media and plated. Finally, individual yeast colonies were picked for Sanger sequencing.

Human antibody variable gene transcripts (VH, Vκ, Vλ) were amplified by RT-PCR using SuperScript IV enzyme (Thermo Scientific Cat# 18090050) followed by nested PCR using HotStarTaq Plus DNA Polymerase (Qiagen Cat# 203646) and a mixture of IgM-, IgD-, IgA-, and IgG-specific constant-region primers as previously described by Wec et al. Science 2020 and included in Supplementary Data 2. The primers used in the second round of nested PCR contained 40 base pairs of 5′ and 3′ homology with linearized yeast expression vectors to allow cloning by homologous recombination. Amplified transcripts were transformed into S. cerevisiae using the lithium acetate method for chemical transformation^{49 (link)}. Per transformation reaction, yeast cells (1 × 10⁷) were incubated with a mixture of 240 μl of polyethylene glycol (PEG) 3350 (50% w/v) (Sigma-Aldrich, Cat# 202444), 36 μl of 1 M lithium acetate (Sigma-Aldrich, Cat# 517992), 10 μl of denatured salmon sperm DNA (Invitrogen, Cat# 15632011), 67 μl sterile water, 200 ng of each of the digested vectors and 10 μl each of unpurified VH and VL amplified PCR product at 42 °C for 45 min. Yeast were then washed twice with sterile water, recovered in selective media, and plated for Sanger sequencing.

Antibody variable gene fragments (VH, Vk, and Vλ) were amplified by RT-PCR as described previously in ref. ^{37 (link)}. Briefly, cDNA was synthesized using randomized hexamers and SuperScript IV enzyme (Thermo Fisher Scientific). cDNA was subsequently amplified by two rounds of nested PCRs, with the second cycle of nested PCR adding 40 base pairs of flanking DNA homologous to restriction enzyme-digested S. cerevisiae expression vectors to enable homologous recombination during transformation. PCR-amplified variable gene DNA was mixed with expression vectors and chemically transformed into competent yeast cells via the lithium acetate method^{38 (link)}. Yeast were plated on selective amino acid drop-out agar plates and individual yeast colonies were picked for sequencing and recombinant antibody expression.