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One step reverse transcription and amplification kit

Manufactured by Qiagen
Sourced in United States

The One-Step reverse transcription and amplification kit is a laboratory product that enables the combined reverse transcription and amplification of RNA in a single reaction. It facilitates the detection and quantification of RNA targets.

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2 protocols using one step reverse transcription and amplification kit

1

Mouse TCR Library Preparation

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For TCR library preparation, we used the commercially available iRepertoire platform (iRepertoire, Huntsville, AL, USA) for nested amplicon arm-PCR of the CDR3 of the mouse TCR β-chains and addition of adaptors for Illumina platform sequencing. Reverse transcription of 300–500 ng of RNA was conducted with a One-Step reverse transcription and amplification kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. The PCR product was purified using Ampure XP magnetic beads (Agencourt/Beckman Coulter, Brea, CA, USA), and secondary amplification of the resulting product was performed (GoTaq PCR Kit, Promega, Madison, WI), allowing addition of Illumina adapter sequences (manufacturer’s protocol). Libraries were purified with Ampure XP magnetic beads and sequenced using Illumina MiSeq 150 nt paired-end read-length. The TCR CDR3 sequences were extracted from the raw sequencing data by iRepertoire.
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2

Murine TCR beta sequencing protocol

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TCR sequencing was performed using the Mouse TCR beta, Illumina, V-C genes kit from iRepertoire, Huntsville, AL. In brief, 500 ng of RNA was reverse transcribed using a One-Step reverse transcription and amplification kit (Qiagen). The PCR product was purified using Ampure XP magnetic beads (Agencourt), and amplified again (TopTaq PCR Kit, Qiagen), to add adaptor sequences. Libraries were purified with Ampure XP magnetic beads and sequenced using Illumina MiSeq 150 nt paired-end read-length. The TCR CDR3 sequences were extracted from the raw sequencing data by iRepertoire. Briefly, raw paired-end fastq files were first demultiplexed based on barcode and merged reads were mapped using a Smith-Waterman algorithm to germline V, D, J and C reference sequences from the IMGT web site (http://www.imgt.org). To define the CDR3 region, the position of CDR3 boundaries of reference sequences from the IMGT database were migrated onto reads through mapping results and the resulting CDR3 regions were extracted and translated into amino acids. Reading frames not containing a stop codon were filtered and error-corrected using iRepertoire proprietary SMART algorithm.
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