Amplified polyclonal cDNA from XNA selections was prepared for deep sequencing by the Illumina Miseq method by appending the bridge-amplification sequences by PCR. Sequencing library generating PCR reactions were performed with OneTaq Hot Start master mix (NEB, USA) with 10 ng/50ul gel-purified polyclonal template DNA (see above), 0.1 μM primers (P5_P2 and P3_Test7-2) and cycling conditions 94°C for 1 min, 10×[94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec], 72°C for 2 min. Sequencing library DNA was purified using a PCR purification kit (Qiagen, Netherlands), then a 12pM sample of pooled libraries plus 20% PhiX control (Illumina, UK) was denatured and sequenced (single-end read, 75 cycles) using a MiSeq reagent kit and instrument (Illumina, UK) according to manufacturer’s instructions. Libraries were barcoded using variants of the P5_P2 primer containing 6nt sequences from the NEXTflex series (Illumina, UK). Data was analysed using the Galaxy server33 (link)-35 (link) and sequences ordered by abundance.
Nextflex series
Manufactured by Illumina
Sourced in United Kingdom
The NEXTflex series is a line of high-performance lab equipment designed for DNA and RNA sequencing applications. The core function of the NEXTflex series is to provide reliable and efficient sample preparation and library construction tools for next-generation sequencing. The product line includes a variety of kits and reagents tailored to specific sequencing needs.
Automatically generated - may contain errors
2 protocols using nextflex series
1
Illumina-based deep sequencing of XNA selections
2
Illumina-based deep sequencing of XNA selections
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Amplified polyclonal cDNA from XNA selections was prepared for deep sequencing by the Illumina Miseq method by appending the bridge-amplification sequences by PCR. Sequencing library generating PCR reactions were performed with OneTaq Hot Start master mix (NEB, USA) with 10 ng/50ul gel-purified polyclonal template DNA (see above), 0.1 μM primers (P5_P2 and P3_Test7-2) and cycling conditions 94°C for 1 min, 10×[94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec], 72°C for 2 min. Sequencing library DNA was purified using a PCR purification kit (Qiagen, Netherlands), then a 12pM sample of pooled libraries plus 20% PhiX control (Illumina, UK) was denatured and sequenced (single-end read, 75 cycles) using a MiSeq reagent kit and instrument (Illumina, UK) according to manufacturer’s instructions. Libraries were barcoded using variants of the P5_P2 primer containing 6nt sequences from the NEXTflex series (Illumina, UK). Data was analysed using the Galaxy server33 (link)-35 (link) and sequences ordered by abundance.
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