Targeted PCR was used for amplification of TRB sequences from genomic DNA, using a cocktail of forward primers specific for framework region 2 (FR2) sequences of 23 TRBV subgroups (gene families), and 13 TRBJ region reverse primers adapted from the BIOMED2 primer series [22 (link)]. Amplicons were purified using the Agencourt AMPure XP beads system (Beckman Coulter, Inc.). Second-round PCRs to generate the sequencing libraries were carried out using NexteraXT Index Primers S5XX and N7XX. Libraries were sequenced using an Illumina MiSeq in the Human Immunology Core Facility at the University of Pennsylvania. 2 × 300 bp paired end kits were used for all experiments (Illumina MiSeq Reagent Kit v3, 600 cycle, Illumina Inc., Cat. No. MS-102-3003).
Agencourt ampure xp beads system
Manufactured by Beckman Coulter
The Agencourt AMPure XP beads system is a magnetic bead-based purification solution used for the separation and purification of nucleic acids, including DNA and RNA, from various sample types. The system utilizes carboxylate-modified magnetic beads to selectively bind and isolate nucleic acids, allowing for efficient removal of contaminants and impurities.
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2 protocols using agencourt ampure xp beads system
1
Amplification and Sequencing of TRB Repertoire
2
HPV DNA Amplification and Illumina Sequencing
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HPV DNA was amplified by PCR using modified GP5+/GP6+ primer pairs extended with overhang adapter sequences required to bind the Illumina® indexes and sequencing [19 (link)]. PCR was performed in 50 μL reaction mixtures containing 1X High-Fidelity Platinum buffer, 3.5 mM MgCl2, 0.6 μM forward and reverse primers and 1U of proofreading Taq polymerase (Platinum™ Taq DNA Polymerase, Thermo Fisher®). The cycling protocol consisted of an activation step (15 min at 95 °C), 21 cycles (1 min at 94 °C and 2 min at 50 °C, decreased by 0.5 °C per cycle to 40 °C and 1.5 min at 72 °C), 24 cycles (1 min at 94 °C, 2 min at 40 °C, 1.5 min at 72 °C), and a final elongation step (4 min at 72 °C) [20 (link)]. Each PCR product was dual-indexed using the KAPA HiFi HotStart Uracil + ReadyMix® (Roche Diagnostics®). The Agencourt Ampure XP beads system (Beckman Coulter®) was used to purify the DNA libraries. The concentrations of the DNA libraries were normalised prior to pooling and sequencing to ensure equal representation of each sample. A PhiX Control (Illumina®) spike-in at 5% was used as an internal control to monitor sequencing quality. The combined library and the PhiX Controls were loaded at 8 pM final concentration on MiSeq using the v3 reagent kit and sequenced by Illumina MiSeq 2 × 300-bp paired-end sequencing.
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