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Miseq 150 nt

Manufactured by Illumina

The MiSeq 150 nt is a next-generation sequencing instrument manufactured by Illumina. It is designed to perform sequencing of nucleic acid samples and generate reads up to 150 nucleotides in length.

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2 protocols using miseq 150 nt

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

Extraction and Sequencing of Total RNA

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Total RNA was extracted using TRIZOL from wildtype Dictyostelium discoideum cells (orfj) grown axenically in 1.5xHL+FM medium. RNA was treated with DNase I, digested twice with RNase R, combined with similarly treated Neurospora crassa RNA, and then processed to a barcoded paired-end Illumina sequencing library using 1/4th of a normal ScriptSeq reaction (Epicentre). This library was included as 1/15th of a pool of libraries for a single Illumina MiSeq 150 nt paired-end run.
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