In vitro-purified RNA (1 μg) was reverse transcribed by using 200 U SuperScript II under standard buffer conditions (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 10 mM dithiothreitol [DTT], 6 mM MnCl2, and 0.5 mM deoxynucleoside triphosphate [dNTP] mix) with random hexamers (LifeTech). Second strand synthesis was performed by using NEBNext Ultra II nondirectional second strand synthesis module (New England BioLabs [NEB]). Double-stranded cDNA was purified with Monarch DNA cleanup kits (NEB) and diluted to 0.2 ng/μL. Sequencing libraries were generated by using a Nextera XT DNA library preparation kit (Illumina) according to the manufacturer’s instructions. Libraries were quantified by using a Qubit instrument (Life Technologies) and bioanalyzer (Agilent) and sequenced on a NextSeq 500/550 system (Illumina).
Nebnext ultra 2 nondirectional second strand synthesis module
Manufactured by New England Biolabs
The NEBNext Ultra II nondirectional second strand synthesis module is a laboratory instrument designed for the synthesis of complementary DNA (cDNA) strands from RNA templates. The module facilitates the conversion of single-stranded RNA into double-stranded cDNA, which is a crucial step in various molecular biology workflows, such as gene expression analysis and next-generation sequencing library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Qubit instrument, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 550 system, by Illumina (1 mentions)
Monarch dna clean up kits, by New England Biolabs (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Nextseq 500 instrument, by Illumina (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
2 protocols using nebnext ultra 2 nondirectional second strand synthesis module
1
RNA-Seq Library Preparation Protocol
2
Nextera XT Library Generation from cDNA
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Nextera XT libraries were built from cDNA obtained in each MRT reaction (Mg or Mn). First, second strand synthesis was performed with the NEBNext Ultra II Non-Directional Second Strand Synthesis Module (NEB E6111S) following the manufacturer’s instructions. The dsDNA was then purified with the Monarch PCR and DNA cleanup kit (T1030S) and quantified on an Invitrogen Qubit fluorometer. Paired-end sequencing libraries were generated from each dsDNA sample using the Nextera XT DNA Library Preparation Kit (Illumina FC-131–1024) exactly as recommended by the manufacturer. Final libraries were purified with AMPure XP beads (Beckman Coulter A63880) using a 1:1 beads/sample ratio, quantified on an Invitrogen Qubit fluorometer and analyzed on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq 500 instrument.
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