Single-cell BCR-seq of human: adult male PBMC
RNA was reverse-transcribed into cDNA using a biotinylated oligo dT primer. An adaptor sequence was added to the 5' end of all cDNA, which contains the Illumina P7 universal priming site and a 17-nucleotide unique molecular identifier (UMI). Products were purified using streptavidin-coated magnetic beads followed by a primary PCR reaction using a pool of primers targeting the IGHA, IGHD, IGHE, IGHG, and IGHM regions, as well as a sample-indexed Illumina P7C7 primer. The immunoglobulin-specific primers contained tails corresponding to the Illumina P5 sequence. PCR products were then purified using AMPure XP beads. A secondary PCR was then performed to add the Illumina C5 clustering sequence to the end of the molecule containing the constant region. The number of secondary PCR cycles was tailored to each sample to avoid entering plateau phase, as judged by a prior quantitative PCR analysis. Final products were purified, quantified with Agilent Tapestation and pooled in equimolar proportions, followed by high-throughput paired-end sequencing on the Illumina MiSeq platform. For sequencing, the Illumina 600 cycle kit was used with the modifications that 325 cycles was used for read 1, 6 cycles for the index reads, 300 cycles for read 2 and a 20% PhiX spike-in to increase sequence diversity.
SUB9071015
NANA01_NBV_PBMC-2