Strand-specific RNA-seq libraries were constructed using the NEB Ultra II RNA-seq library kit (NEB #E7765) according to manufacturer's protocol with fragmentation in first-strand buffer at 94°C for 15 min. Following first and second strand synthesis, DNA was purified with 1.8× AmpureXP beads (Beckman #A63880), end repaired, then ligated to sequencing adaptors diluted 1:5. Ligated DNA was purified with 0.9× AmpureXP beads and PCR amplified for 8 cycles, then purified again with 0.9× AmpureXP beads. Libraries were verified by Qubit dsDNA high sensitivity (Invitrogen #Q32851) and Fragment Analyzer prior to multiplexed paired-end sequencing on an Illumina NextSeq 500 at the Health Sciences Sequencing Core at Children's Hospital of Pittsburgh.
Neb ultra 2 rna seq library kit
Manufactured by New England Biolabs
The NEB Ultra II RNA-seq library kit is a product designed for the preparation of RNA sequencing libraries. It provides a streamlined workflow for generating high-quality libraries from total RNA samples.
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2 protocols using neb ultra 2 rna seq library kit
1
Strand-specific RNA-seq Library Prep
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Strand-specific RNA-seq Library Preparation
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Strand-specific RNA-seq libraries were constructed using the NEB Ultra II RNA-seq library kit (NEB #E7765) according to manufacturer's protocol with fragmentation in first-strand buffer at 94°C for 15 minutes. Following first and second strand synthesis, DNA was purified with 1.8X AmpureXP beads (Beckman #A63880), end repaired, then ligated to sequencing adaptors diluted 1:5. Ligated DNA was purified with 0.9X AmpureXP beads and PCR amplified for 8 cycles, then purified again with 0.9X AmpureXP beads. Libraries were verified by Qubit dsDNA high sensitivity (Invitrogen #Q32851) and Fragment Analyzer prior to multiplexed sequencing (paired end 38/37bp) on an Illumina NextSeq 500 at the Health Sciences Sequencing Core at Children's Hospital of Pittsburgh.
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