P5
Following the manufacturers protocols, sorted plasmablasts were prepared with the Chromium Next GEM Single Cell 5 Reagent Kit v2 (10x Genomics, Pleasanton, CA, US; 1000244), combined with barcoded Gel Beads (10x Genomics, Pleasanton, CA, US; 1000267), and partitioned into Gel Beads-in-emulsion (GEMs) using a Chromium X controller and the Chromium Next GEM Chip K Single Cell Kit (10x Genomics, Pleasanton, CA, US; 1000286). Following GEM generation, the GEMs underwent reverse transcription to produce cDNA containing an Illumina TruSeq Read 1 sequence, unique molecular identifier (UMI), template switch oligo (TSO), and 10x barcode. The resulting cDNA was then purified using Dynabeads MyOne SILANE (10x Genomics, Pleasanton, CA, US; 2000048) and amplified with reagents from the Chromium Next GEM Single Cell 5 Reagent Kit v2 (10x Genomics, Pleasanton, CA, US; 1000244) to facilitate B cell receptor (BCR) amplification using a BCR Amplification Kit (10x Genomics, Pleasanton, CA, US; 1000253). Utilizing a Library Construction Kit (10x Genomics, Pleasanton, CA, US; 1000190), the BCR transcripts subsequently went through fragmentation, end-repair, A-tailing, adaptor ligation, and sample index PCR to generate V(D)J libraries containing standard Illumina primers, P5 and P7, dual indices, i5 and i7, and an Illumina TruSeq Read 2 sequence. Following cDNA amplification, BCR amplification, and library construction, samples were purified using SRPIselect (Beckman Coulter, Brea, CA, US; B23317) and quantified on an Agilent Tape Station 4150. Prior to sequencing, the size (bp) of the V(D)J libraries was quantified on an Agilent TapeStation 4150 using a D5000 ScreenTape and Reagents (Agilent Technologies, Santa Clara, CA, US; 5067-5588, 5067-5589), while the concentration (ng/uL) was measured on a Qubit 4 using a Qubit 1X dsDNA High Sensitivity (HS) Assay Kit (Invitrogen, Waltham, MA, US; Q33231). The V(D)J libraries were then denatured and diluted using a MiSeq Reagent Kit v3 (150-cycle) (Illumina, San Diego, CA, US; MS-102-3001) and sequenced on an lllumina MiSeq. Following sequencing, FASTQ files were converted from raw BCL files using the Illumina Basespace platform and processed for data analysis on the 10x Genomics Cloud.