Oligo dt 25 magnetic beads

PBMCs were isolated by centrifuging 20 mL of fresh blood overlaid with the same volume of 100% Ficoll (GE Healthcare) for 40 min at 400 × g. Memory B cell and the plasmablast fraction were separated using MACS isolation kit (Miltenyi Biotec), according to the manufacturer’s instructions. Approximately 5 × 10⁶ cells were washed with PBS and resuspended in 500 μL of FACS buffer (0.5% BSA and 0.05% azide in PBS). Cells were then stained with anti-human CD19-Brilliant Violet 421, anti-human IgG-APC and the recombinant ECD of the α-subunit of nAChR conjugated with PE to detect memory B cells [33 (link)–35 (link)]; and anti-human CD27-APC, CD19-FITC, and CD38-Brilliant Violet 421 for plasmablasts [36 (link),37 (link)]. After 30 min of incubation on ice, cells were washed twice with FACS buffer and resuspended in 500 μL of the same buffer containing diluted 7-Amino-Actinomycin D (7-AAD) (BD Biosciences) prior to cell sorting.
Single CD19 ⁺⁺ and IgG⁺⁺ memory B cells with or without antigen⁺⁺ (recombinant ECD of the α-subunit of human nAChR), as well as single CD27⁺⁺, CD19⁺⁺, and CD38⁺⁺ plasmablasts, were sorted into each well of U-bottom 96-well plates containing 10 μL of cell lysis solution with 10 μg oligo-(dT)₂₅ magnetic beads (Thermo Fisher Scientific) after excluding debris and dead cells based on scatter signals, 7-AAD fluorescence and doublet cells signals.

Global transcriptome analysis was profiled using RNA isolated from embryos with or without ABA treatment, using a method similar to previously described^{63 (link)}. For each sample, equal quantities of high-quality RNA from 12 fruits were pooled for cDNA library construction. Briefly, 2 µg DNA-free total RNA from each sample were purified via oligo(dT)25 magnetic beads (Invitrogen), and then fragmented in 5 × first strand buffer (Invitrogen) at 94 °C for 2 min. The cleaved RNA fragments were reverse-transcribed into the first-strand cDNAs by 3′ and 5′ adaptors, followed by 18 cycles of PCR amplification with Illumina adapters with Phire II (Thermo Fisher Scientific). The library was run on an agarose gel, and a 300 to 500 nt band was excised and purified by using QIAquick gel extraction kit (Qiagen). The final PAT-seq libraries were quality checked by Qubit and Agilent Bioanalyzer 2100 before Illumina HiSeq 2500 sequencing was performed at an in-house facility at the College of the Environment and Ecology, Xiamen University. All RNA-seq read files are available from the NCBI Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov) (accession number SRP107989).

Total RNA isolation, synthesis of a cDNA library attached to oligo(dT)₂₅ magnetic beads (Invitrogen, Carlsbad, CA, USA), and rapid amplification of cDNA ends (RACE PCR) was performed as described previously [18] (link). The resulting PCR products were cloned into pJet1.2 and sequenced by Eurofins Genomics (Ebersberg, Germany). Genomic DNA was isolated according to an established protocol [19] (link). For the generation of C-terminal YFP fusions genes the full length gene was amplified from genomic DNA using oligonucleotide primers that introduced either a SnaBI or EcoRV restriction site at the 5′-end and an XbaI restriction site at the 3′-end of the gene. The genes were amplified using Phusion DNA polymerase (Thermo Fisher Scientific), and the resulting PCR products were cloned into pJet1.2 and sequenced. The pJet1.2/AC genes were subsequently digested with the appropriate restriction enzyme and cloned into the SnaBI/XbaI site of pPhat1/YFP+fcpA/nat. Due to the apparent instability of the AC4076-YFP fusion gene it was necessary to use SURE2 E. coli cells (Agilent; Waldbronn, Germany) for its cloning. All other cloning procedures were performed using DH5α.