Zika_Pt1_Heavy_m2
NGS of homo sapien: antibody heavy chain library (month2)
An improved version of the 5-RACE PCR protocol was used for sample preparation. Total RNA was extracted from 1~5 million PBMCs into 30ml of water with RNeasy Mini Kits (Qiagen). For unbiased repertoire analysis, 5-RACE was performed with SMARTer RACE cDNA Amplification Kit (Clontech). 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 5-RACE primer, 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 5-RACE PCR products at ~600 bp were gel purified (Qiagen). Antibody NGS was performed on the Ion GeneStudio S5 system. Briefly, the antibody heavy and light (k and l) 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. The k and l chain libraries at each time point were mixed at a ratio of 1:1 prior to library pooling and chip loading. 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.
Zika_Pt1_Heavy_m2
SUB6138360
Zika_Pt1_Heavy_m2