title: NGS of homo sapien: ZK2B10-specific heavy chain library (month2)
design: Total RNA was extracted from 1~5 million PBMCs into 30ml of water with RNeasy Mini Kits (Qiagen). For ZK2B10 gene-specific lineage analysis, reverse transcription (RT) was performed with SuperScript III (Life Technologies) and oligo (dT). The cDNA was purified and eluted in 20ul of elution buffer (NucleoSpin PCR Clean-up Kit, Clontech). The immunoglobulin PCRs were set up with Platinum Taq High-Fidelity DNA Polymerase (Life Technologies) in a total volume of 50 l, with 5 l of cDNA as template, 1 l of gene-specific forward primers, and 1 l of 10 M reverse primer. To facilitate deep sequencing on the Ion GeneStudio S5 system, the forward primers contained a P1 adaptor, while the reverse primer contained an A adaptor and an Ion XpressTM barcode (Life Technologies) to differentiate the libraries from various time points. A total of 25 cycles of PCRs were performed and the gene-specific PCR products at ~500 bp were gel purified (Qiagen). A degenerate primer (SAGGTGCAGCTGGTGCAGTCTGG) was used as the forward gene-specific primer to cover potential variations at the 5-end of ZK2B10 transcripts. Antibody NGS was performed on the Ion GeneStudio S5 system. Briefly, the antibody heavy chain libraries were quantitated using Qubit 2.0 Fluorometer with Qubit dsDNA HS Assay Kits. Equal amounts of the heavy chain libraries from various time points were mixed and loaded onto an Ion 530 chip to increase the sequencing depth and to eliminate run-to-run variation. Template preparation and (Ion 530) chip loading was performed on the Ion Chef system using Ion 530 Ext Kits, followed by S5 sequencing with the default settings. Raw data was processed without 3-end trimming in base calling.
library_name: Zika_Pt1_Heavy_focus_m2