IMPACT-Seq(64 (link)) was undertaken on 5×106 cells transfected with 3’ biotinylated miRIDIAN miR-96 mimic and scramble (50 nM, 24h and 48h) (Dharmacon, Inc.) using Lipofectamine 2000. RNA was then extracted from washed beads using Trizol LS and treated with T4 Polynucleotide Kinase (New England Biolabs) to obtain 5’ phosphate ends for subsequent ligations and passed through NucAway columns (Ambion) to remove RNAs <20 nt in length. Small RNA libraries were generated using NEBNext Multiplex Small RNA Library Prep (New England Biolabs) following the manufacturer’s recommendations for 100ng input material. PCR-amplified small RNA libraries were size selected to 130–200 bp fragments. Libraries characterized on an Agilent Tape Station on a DNA1000 High-Sensitivity Screen Tape and quantified by Qubit were pooled and sequenced at 75 bp read length on the MiSeq platform.
Nebnext multiplex small rna library prep
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Multiplex Small RNA Library Prep is a laboratory equipment product designed for the preparation of small RNA libraries for next-generation sequencing. It provides a streamlined workflow for the isolation, ligation, and amplification of small RNA molecules from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Nucaway columns, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Horizon Discovery (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Mid output kit, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
High sensitivity dna kit, by Agilent Technologies (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
5 protocols using nebnext multiplex small rna library prep
1
Identification of miR-96 Targets via IMPACT-Seq
2
Small RNA Sequencing of ISV-infected Mosquitoes
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Three pools of 20 ovaries, testes or carcasses from the ISV-infected mosquitoes were combined for the generation of small RNA libraries. 19-33 nt long RNAs were cut and extracted from a 15% acrylamide/bis-acrylamide (37.5:1), 7M urea gel and the purified RNAs were subjected to small RNA library preparation using the NEBNext Multiplex Small RNA Library Prep (New England Biolabs, Ipswich, MA, USA) kit with a 3’ adaptor from Integrated DNA Technologies (Coralville, IA, USA) and in-house designed indexed primers. Libraries diluted to 4 nM were sequenced with the NextSeq 500 High Output Kit v2 (75 cycles) using a NextSeq 500 (Illumina, San Diego, CA, USA). Small RNA libraries have been submitted to the NCBI sequence read archive (SRA) under BioProject PRJNA587399.
3
Sequencing of Small RNAs from Infected Flies
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For each time point of infection analyzed, total RNA was extracted from 15 flies. For each sample, 19-31 nt long RNAs were cut and extracted from a 15% acrylamide/bis-acrylamide (37.5:1), 7M urea gel and the purified RNAs were subjected to small RNA library preparation using the NEBNext Multiplex Small RNA Library Prep (New England Biolabs) kit with a 3′ adaptor from Integrated DNA Technologies (IDT) and in-house designed indexed primers. Libraries diluted to 4 nM were sequenced with the NextSeq 500 High Output Kit v2 using a NextSeq 500. Reads were analyzed using in-house Perl scripts.
4
Transcriptome and Small RNA Profiling
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Total RNA from 5 to 10 individual larvae were extracted using TRIzol (Invitrogen) following the recommended protocol. RNA integrity was verified using the 2100 Bioanalyzer system (Agilent). mRNA libraries were constructed using the kits NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext UltraTM II Directional RNA Library Prep Kit for Illumina (New England BioLabs) following the manufacturer protocol. Small RNA libraries were built using the kit NEBNext Multiplex Small RNA Library Prep (New England BioLabs) following the manufacturer protocol, except for the 5’ RNA adaptor that was modified to include 6 nt at its 5’ extremity. These extra nucleotides are sequenced along with the small RNA ligated and removed at the bioinformatic analysis. Indexed libraries were pooled and sequenced at the GenomEast sequencing platform at the Institut de Génétique et de Biologie Moléculaire et Cellulaire of Strasbourg, France.
5
NextSeq 500 Small RNA Sequencing
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Processing, cDNA synthesis, library preparation, and sequencing were performed by the core unit system medicine at the University of Würzburg. The RNA quality was checked using a 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Here, cDNA libraries suitable for sequencing were prepared from 500 ng of total RNA, fragmented for 2 min and 45 s at 94 °C and treated with T4 PNK for phosphorylation/dephosphorylation and RppH for decapping, followed by NEBNext® Multiplex Small RNA Library Prep. (New England Biolabs, Ipswich, MA, USA) without rRNA depletion. The number of the PCR cycles was determined as 13 by qPCR and the elongation time was set to 30 s. Libraries were quantified by a QubitTM dsDNA HS Assay Kit 3.0 fluometer (ThermoFisher, Waltham, MA, USA) and quality was checked using a 2100 Bioanalyzer with a high-sensitivity DNA kit (Agilent Technologies) before pooling. Sequencing of pooled libraries, spiked with 5% PhiX control library, was performed in single-end mode with 150 nt read length on the NextSeq 500 platform (Illumina, San Diego, CA, USA) with a Mid Output Kit (Illumina).
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