GSM5101276: nanoMouse2_Spike_after; Mus musculus; OTHER
Diversity: Llama nanobody phage library was constructed as described before (PMID:24577359) with some modifications. Briefly, 300 ml of whole blood was collected from llama and peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque plus (GE Healthcare, 17-1440-03) according to manufacturer's instruction. Total RNA e  xtracted from PBMC was used as starting material for RT-PCR to amplify the variable domains of immunoglobulin heavy chain (VHH) by two steps of PCR reactions. 50ug of RNA was reverse transcribed to cDNA with random hexamers and 2.5ul of cDNA (corresponding to about 0.9ug of original RNA) was used for first round PCR with gene-specific primers CALL001 and CALL002. The PCR reaction was repeated in 12 individual tubes with cDNA added into reactions separately. PCR fragment of about 700 bp long was gel purified and used as template (30 ng for each reaction, repeated in 12 individual tubes) for second round PCR with nested primers VHH-Back and VHH-For to amplify nanobody-encoding genes. PCR product from individual reactions werepooled, gel purified. Nanobody fragments and pMES4 phagmid were digested with PstI-HF and BstEII-HF restriction enzymes (NEB R3140L, R3162L) and ligated (1ug and 2ug respectively) with T4 ligase at 16 °C overnight. Ligation product was column purified (into 12ul of H2O) and electroporated into 360ul of TG1 cells. After 1 hour of recovery in 37 °C in shaker incubator, cells were plated on 6 of 245mm x 245mm dish (Thermo Fisher Scientific,431301) containing 2-YT Agar supplemented with 100ug/ml carbenicillin and 2% (wt/vol) glucose. Plates were placed in 37 °C bacteria incubator overnight and then bacteria scaped off plates. Cells were infected with VSCM13 helper phage (Agilent Technologies, 200251) and nanobody phage library preprepared as described before (PMID: 24577359). Nanomouse nanobody phage libraries were constructed the same way as nanomouse VHH(D)J region phagemid library construction with some modifications. Briefly, total RNA was extracted from splenic cells, bone marrow (femur and tibia) cells and PBMC of immunized mice and processed separately until TG1 cell electroporation step. RNA from splenic cells, bone marrow and PBMC (50ug, 50ug and all respectively) were reversed transcribed to cDNA with Ighg1 CH2-specific primer in separate tubes. 2ul of cDNA was used as template for BCR variable domains amplification (12 reactions each), using unmodified  TSO and Ighg1 CH2-specific oligonucleotide as primers. Second PCR was repeated in 12 reactions with 30 ng of the first-step PCR product as template 30 FR1 and 4 JH oligonucleotide mix as primers. PCR products were gel purified, digested with SfiI and ligated with pMES4 (200 ng and 400 ng respectively). Ligation products from splenic cells, bone marrow and PBMC samples were pooled and column purified (into 12ul of H2O) and electroporated into 360ul of TG1 cells and phage libraries prepared as described above.  VHH usage analysis: BCR VHH(D)J region phage library of unimmunized nanomouse was constructed and deep sequenced by Illumina Miseq for VHH usage analysis. Briefly, total RNA was extracted from nanomouse splenic cells by Trizol Reagent (Thermo Fisher Scientific,15596026) and reverse transcribed to cDNA with SuperScript™ III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, 18080400) according to manufacturer's instructions with some modifications.10ug of total RNA was denatured and annealed with gene specific primer corresponding to CH2 of Ighm gene. After elongation at 50 °C for 50 minutes, template switching oligonucleotide (TSO, 3'-propyl modified) linker was added to the 3' end of the first strand cDNA with 90 minutes incubation at 42 °C as described before (Fernando Lopes Pinto et al. 2010, Anal Biochem. PMID: 19837043). Reaction was inactivated at 85 °C for 5 minutes and 2ul of cDNA was used as template for BCR variable domains amplification by two-step PCR with CloneAmp HiFi PCR Premix (Takara, 639298). For the first-step PCR, unmodified TSO and Ighm CH2-specific oligonucleotide were used as primers. 30 ng of the first-step PCR product was used as template for second-step nested PCR with a primer mix of 30 forward primers corresponding to framework (FR1) of 30 VHH genes and 4 reverse primers corresponding to JH1~JH4 of Igh gene. pMES4 phagemid was amplified with primers to introduce two different SfiI sites on two ends. Both VHH(D)J and pMES4 fragments were digested with restriction enzyme SfiI (NEB, R0123L) and ligated (100 and 200 ng respectively) with T4 ligase (NEB, M0202L) at 16 °C overnight. Ligation product was purified with DNA Clean & Concentrator (Zymo Research, D4014) and eluted into 12ul of H2O. 3ul of DNA was electroporated into 60ul of TG1 cells (Lucigen, 60502-2) in 1.0 mm cuvette (HARVARD Apparatus, 450134) with BTX electroporation system ECM 630 at the setting of 25uF, 200 Ohms, 1600 Volts. After 1 hour of recovery in 37 °C in shaker incubator, TG1 cells were plated on 5 of 10-cm LB Agar plates supplemented with 100ug/ml carbenicillin (KD Medical, BPL-2400). Plates were placed in 37 °C bacteria incubator overnight and then bacteria scaped off plates and phagemid library DNA extracted with Zymo Plasmid Miniprep kit (Zymo Research, D4054). Phagemid DNA extracted from TG1 cell libraries was used as starting material for constructing MiSeq libraries for both VHH usage and nanobody diversity analysis. Briefly, 1.2 ug of phagemid DNA was used as template and VHH(D)J inserts were amplified by two primers recognizing pMES4 backbone with CloneAmp HiFi PCR Premix (Takara, 639298) in 50 ul reaction volume (9 cycles). To avoid MiSeq failure due to low complexity at initial cycles and to enable multiplex sequencing, 1-9 nt long staggers were introduced into forward primers. Without purification, 5 ul of the first PCR product was used as template for a second PCR (9 cycles) to add Illumina P5 and P7 primers on DNA ends. PCR product was then loaded onto 2% agarose gel and ~580 bp size band was purified with Zymoclean Gel DNA Recovery Kit (Zymo Research, D4002). DNA concentration was determined by Qubit 4  Fluorometer (Thermo Fisher Scientific, Q33238) and average DNA size was determined by TapeStation 4150 (Agilent). DNA was then adjusted to 2 nM in elution buffer containing 0.1% Tween-20. For unimmunized nanomice VHH(D)J library, DNA (2nM) from 3 mice were mix at 1:1:1 ratio and loaded for MiSeq run. For immunized llama and nanomice nanobody diversity analysis, DNA (2nM) of pre-selection and post-selection library were mixed at 10:1 ratio first and then samples from individual animals were pooled at 1:1 ratio before loading for MiSeq Sequencing (Illumina). 270 cycles of paired-end sequencing data were acquired  for VHH usage analysis and 300 cycles of paired-end sequencing data were acquired for nanobody diversity analysis.