| Library |
| Library name |
Construction protocol |
Strategy |
Source |
Selection |
Layout |
|
VDJ-Seq analysis of the Igh locus was conducted as previously described with the following modifications: Genomic DNA was extracted from bone marrow cells or spleen cells (~10^6 cells) using the Qiagen DNeasy Blood & Tissue Kit (QIAGEN, 69504) according to the manufacturer’s instructions; DNA was fragmented, end-repaired, and adapter-ligated using the NEBNext Ultra II FS DNA Library Prep Kit (E7805, E7805) with adapter sequences; The ligation products were purified using 1X AMPure XP beads (Beckman Coulter, A63881) following the manufacturer’s instructions to remove unligated adapters; Single-stranded DNA was prepared by primer extension using biotin-labeled JH-specific primers, and these single-stranded DNA products were captured using Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific, 65601); PCR amplification was performed with nested JH-specific primers and adapter-ligated primers, with Illumina sequencing adapters containing multiplex library indices added during the final PCR step; DNA quantification was performed using Qubit (Thermo Fisher Scientific), and library size distribution and quality were assessed with the Agilent 2100 Bioanalyzer, ensuring library sizes ranged between 200 and 1000 bp with no significant peaks below 200 bp;Paired-end 250 bp sequencing was conducted on a NovaSeq 6000 platform according to the manufacturer’s instructions; After demultiplexing, adapter removal, and Q30 read filtering, the data were aligned using a custom library containing all V, D, and J genes of the synthetic immunoglobulin locus in MIXCR (v4.7.0), and the utilization of each gene along with the dimensionality reduction of VDJ products was analyzed using a custom pipeline. |
BCR-Seq |
GENOMIC |
PCR |
PAIRED
|
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