title: GSM6339423: Hashed PacBio scBCR library 1; Homo sapiens; RNA-Seq
library_construction_protocol: Peripheral blood was collected in heparin tubes (BD, #367874) and processed within 24 hours. Blood was spun at 1,500 rpm for 10 min. The plasma layer was collected and stored at -80°C. PBMCs were separated by Ficoll-Paque Plus density gradient centrifugation (Sigma-Aldrich, #GE17-1440-02) using Leucosep tube (Grainer Bio-One, #227288) following the manufacturer's instructions. After the isolation, the PBMCs were counted and resuspended in 500 ul of 5% AB serum in RPMI and 500 ul of 20% DMSO in AB serum at 0.5 – 10 x 106 PBMCs per cryovial and cryopreserved. 10X Genomics libraries:B cells were isolated from 17 million to 100 million PBMC from 5 peanut allergic children with sIgE>100 and 3 non-allergic children by EasySepTM Human B cell Isolation Kit (STEMCELL technologies, #17954). Hash tagged antibodies were added to 3 million B cells from each sample. B cells from 2 peanut allergic subjects were combined in experiment 1. In experiment 2, B cells from 3 non-allergic subjects were combined, and B cells from 3 non-allergic children were combined. The cells were then stained with BV421 anti-human CD19 and FITC anti-human CD27 antibodies and CD19+CD27+ memory B cell populations were sorted on a FACSAria (BD). After confirming cell number and viability, B cells were run on a Chromium 10X controller using 5' chemistry (10X Genomics) in one lane (experiment 1) or two lanes (experiment 2) with an expected recovery rate of 10,000 cells per lane, according to the manufacturer's instructions. Libraries were generated and run on a HiSeq2500 apparatus.  PacBio Libraries: Ara h 2 multimers were generated by associating biotinylated Ara h 2 with PE-labeled streptavidin or AF647-labeled streptavidin. Similarly, detoxified diphtheria toxin (DT) (CRM197, PFENEX Biopharmaceuticals) was used to generate DT multimers. Ara h 2 (or DT) binding B cells were sorted into 96 well plates with lysis buffer containing RNAse inhibitor, dNTPs and oligo dT-VN30 anchored with template switch oligo sequence (Table S4). Plates were flash frozen in dry ice and stored at -80°C. Plates were thawed on ice, then oligo dT was added and annealing was performed at 70°C for 5 min. cDNA synthesis and cDNA amplification was performed with template switching oligo (TSO) using template switching RT enzyme mix (NEB) according to the manufacturer's instruction. The first PCR was performed to amplify BCR heavy and light chains using outer primers targeting human immunoglobulin constant regions (IgG, IgM for heavy chains, kappa and lambda for light chains) and TSO inner primer (Table S4), respectively. After cleaning up the PCR products, BCR heavy and light chains were further amplified with inner primers with plate and row specific ID (5') and column specific ID (3') to enable the identification of BCR sequences derived from each well (Table S4). Afterwards, all PCR products were combined as one library. PacBio sequencing was performed to obtain long amplicon reads to cover 500 to 1000 bp. All PCR reactions were performed using NEBNext UltraTM II Q5 master mix (NEB). BCR sequence data from 10X Genomics scRNA-seq + BCR sequencing began with the filtered V(D)J contigs from Cell Ranger version 3.1. To obtain V and J gene assignments, these contigs were aligned to the IMGT GENE-DB germline reference allele database (obtained 8/3/2019)43. Non-productive heavy and light chain BCR sequences were removed. BCRs were annotated by B cell subtype based on matching their single cell barcodes to the gene expression information. Only cells that had both gene expression and BCR sequence data were retained. In cells with multiple heavy chains, only one heavy chain with the highest unique molecular identifier count was retained. In the event of a tie, the first heavy chain identified was retained. For BCR sequencing data from sorted single cell PacBio sequencing data, adaptor ligation, sequencing reactions, and initial sequence processing were conducted at GENEWIZ, LLC/Azenta US, Inc (South Plainfield, NJ, USA) using standard PacBio circular consensus sequence (CCS) tools and guidelines. Further processing was performed using presto v0.6.244. To remove reads too short to likely contain functional BCRs and associated constant regions, reads shorter than 500bp were removed. After initial processing, PacBio BCR sequence reads were assembled into consensus sequences. As with 10X Genomics BCR sequence data, consensus sequences were aligned to the IMGT GENE-DB database43 to obtain V and J germline annotations. Non-productive heavy and light chain BCR sequences were removed. BCRs from both 10X Genomics and PacBio were processed using the Immcantation suite.