RNA was converted to library using NEBNext Small RNA Library kit (NEB, UK) using the manufacturer's instructions. Library quality was assessed using a High Sensitivity DNA chip on a Bioanalyzer 2100. Library quantification was done using a fluorometer (Qubit 3.0 fluorometer, Invitrogen, USA).
Nebnext small rna library kit
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
The NEBNext Small RNA Library Prep Kit is a library preparation kit designed for the construction of small RNA sequencing libraries. The kit includes reagents and protocols for the enzymatic processing of small RNA samples, including 3' adaptor ligation, reverse transcription, and PCR amplification. The kit is intended for use with various small RNA species, such as microRNAs, small nuclear RNAs, and small nucleolar RNAs.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Antarctic phosphatase, by New England Biolabs (1 mentions)
Rna sample preparation kit v2, by Illumina (1 mentions)
Truseq small rna prep kit, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop 2000, by Agilent Technologies (1 mentions)
Picorna chip, by Agilent Technologies (1 mentions)
Nextseq 550, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Hiseq 1000, by Illumina (1 mentions)
Hiseq 1000 instrument, by Illumina (1 mentions)
12 protocols using nebnext small rna library kit
1
RNA Library Construction and Quantification
2
RNA Sequencing Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA fragments without any gel-purification step were directly 3′-end dephosphorylated using 5 U of Antarctic phosphatase (New England Biolabs, Ipswich, MA, USA) for 30 min at 37 °C. After the inactivation of the phosphatase for 5 min at 70 °C, RNA fragments were phosphorylated at the 5′-end using T4 polynucleotide kinase (PNK) and 1 mM ATP for 1 h at 37 °C. End-repaired RNA fragments were then purified using the RNeasy MinElute Cleanup kit (QIAGEN, Hilden, Germany) according to the manufacturer’s recommendations, except that the volume of 96% ethanol was adjusted for RNA binding. Elution was performed in 10 µL of nuclease-free water. RNA fragments were converted to the library using the NEBNext Small RNA Library kit (ref#E7330S, New England Biolabs; or equivalent from Illumina, San Diego, CA, USA) following the manufacturer’s instructions. DNA library quality was assessed using a High Sensitivity DNA chip on a Bioanalyzer 2100. Library quantification was done using a fluorometer (Qubit 2.0 fluorometer, Invitrogen, Waltham, MA, USA).
The library mix for sequencing was adjusted to obtain ~10–15 millions of raw reads for each library; this number was found to be sufficient for a 5000–8000 nt length reference sequence (end-coverage ~1000×).
The library mix for sequencing was adjusted to obtain ~10–15 millions of raw reads for each library; this number was found to be sufficient for a 5000–8000 nt length reference sequence (end-coverage ~1000×).
3
Small RNA Sequencing of Sclerotinia sclerotiorum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MirVana miRNA Isolation kit (Thermo Fisher Scientific) was used to extract small RNAs from 4-day-old mycelia. NEBNext small RNA Library Kit (NEB, Ipswich, MA, United States) was used to construct the libraries for sequencing. The libraries were barcoded, pooled in a single lane for 50-nt single-end reads sequencing on an HiSeq4000 at the Roy J. Carver Biotechnology Center, UIUC. Three replicates of samples from SsHV2-L virus-infected DK3 as well as four replicates (two virus-infected and two virus-free mutants) each of Δdcl-1, Δdcl-2, Δagl-2, and Δagl-4 samples were sequenced. Adaptors were trimmed by BBMap tools (Bushnell, 2014 ). ShortStack (Axtell, 2013 (link)) was used to identify loci producing sRNAs by clustering. The number of reads aligned to S. sclerotiorum and SsHV2-L genomes were computed using bowtie (Langmead et al., 2009 (link)), and further downstream analysis were performed using in-house Perl and R scripts. tRNA encoding genes were predicted by tRNAscan-SE (Lowe and Chan, 2016 (link)).
4
Small RNA and mRNA Sequencing of Mouse Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated using Trizol (Life Technologies). One hundred nanograms of total RNA was used to generate small RNA libraries using the NEBNext Small RNA Library kit (NEB) or the TruSeq Small RNA Prep Kit (Illumina). miRNA expression was found with MirDeep2 (Friedländer et al. 2012 (link), mm9, miRBase version 21), and differential expression was found with EdgeR (Robinson et al. 2010 (link)). Approximately 100 ng of total RNA was used to generate mRNA-Seq libraries using the RNA Sample Preparation Kit v2 (Illumina). Reads were aligned to the genome with Tophat (Trapnell et al. 2009 (link), mm9), and differential expression was found with CuffDiff (Trapnell et al. 2013 (link)).
5
Sequencing Small RNAs from Fungal Mycelia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small RNAs were extracted from 4-day-old mycelia using mirVana miRNA Isolation kit (ThermoFisher Scientific) following the manufacturer’s protocol. Libraries were prepared using the NEBNext small RNA Library Kit (NEB, Ipswich, MA, USA). The libraries were pooled and sequenced in one lane for 50-nt single-end reads on an Illumina HiSeq4000 at Keck Center, University of Illinois. We sequenced two replicates each of virus-free wtDK3 and Δdcl-1/dcl-2 as well as five replicates each of wtDK3 infected with SsHV2-L and three replicates of wtDK3 infected with SsHADV-1. Demultiplexed reads were removed of the 3′ adaptors by Trimmomatic [21 (link)]. Loci producing sRNAs were identified by ShortStack [22 (link)]. The obtained sequences have been deposited in NCBI (the accession will be available during review).
6
Small RNA Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA fragments were converted to library using NEBNext® Small RNA Library kit (NEB ref E7330S, UK or equivalent from Illumina, USA) following the manufacturer's instructions. DNA library quality was assessed using a High Sensitivity DNA chip on a Bioanalyzer 2100. Library quantification was done using a fluorometer (Qubit 2.0 fluorometer, Invitrogen, USA).
7
RNA-seq and DNA methylome analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The data pertaining to RNA sequencing and methylome have been deposited in NCBI’s Gene Expression Omnibus39 (link) and are accessible through GEO Series accession number GSE117544 . In addition, a secure token has been created to allow the review of the record while it remains in private status and can be requested from the authors.
RNA was extracted from TS and ML cells using Trizol (Fischer Scientific). RNA concentration was measured on Nanodrop 2000 and RNA quality was checked by capillary electrophoresis with PicoRNA chip on Bioanalyzer 2100 (Agilent Les Ulis France). RNA was fragmented and converted to library using NEBNext® Small RNA Library kit (NEB Evry France). DNA library quality was assessed with a High Sensitivity DNA chip on a Bioanalyzer 2100. Library quantification was done using a fluorometer (Fisher Scientific). Libraries were multiplexed and subjected to high-throughput sequencing by using Illumina MiSeq for paired-end read. Data were generated from 3 independent TS cultures and 3 ML cell cultures and analyzed according to the tools detailed in supplemental information.
RNA was extracted from TS and ML cells using Trizol (Fischer Scientific). RNA concentration was measured on Nanodrop 2000 and RNA quality was checked by capillary electrophoresis with PicoRNA chip on Bioanalyzer 2100 (Agilent Les Ulis France). RNA was fragmented and converted to library using NEBNext® Small RNA Library kit (NEB Evry France). DNA library quality was assessed with a High Sensitivity DNA chip on a Bioanalyzer 2100. Library quantification was done using a fluorometer (Fisher Scientific). Libraries were multiplexed and subjected to high-throughput sequencing by using Illumina MiSeq for paired-end read. Data were generated from 3 independent TS cultures and 3 ML cell cultures and analyzed according to the tools detailed in supplemental information.
8
Small RNA Sequencing of Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small RNA libraries were prepared from size fractionation of 10ug of total macrophage RNA on a denaturing polyacrylamide gel to purify 18–35nt small RNAs, and converted into Illumina sequencing libraries with the NEBNext Small RNA Library kit (NEB). Libraries were sequenced on the Illumina NextSeq-550 in the Boston University Microarray and Sequencing Core. We applied long RNAs and small RNAs RNAseq fastq files to a transposon and small RNA genomics analysis pipeline previously developed for mosquitoes (58 (link), 106 (link)) with the mouse transposon consensus sequences loaded instead. Mouse transposon consensus sequences were downloaded from RepBase (107 (link)), and RNA expression levels were normalized internally to each library’s depth by the Reads Per Million counting method.
9
Transcriptome Analysis of Salmonella UV Stress Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Salmonella SL1344 carrying a chromosomal proQ-3xflag allele was grown in 20 mL LB medium to an OD600 of 2.0, after which the cells were washed twice with PBS, once with SPI2 medium, and thereafter diluted 1:50 into fresh SPI2 medium. When the culture reached an OD600 of 0.3, half of the culture was irradiated with UV light (254 nm, 800 mJ/cm2), while the other half was left untreated. Immunoprecipitation, Benzonase treatment, dephosphorylation, radioactive labeling, SDS-PAGE, and RNA purification were carried out as described previously (24 (link)). DNA libraries were prepared using the NEBNext Small RNA Library kit (NEB) according to the manufacturer’s instruction and sequenced on an Illumina NextSeq500 instrument at vertis Biotechnologie AG (Freising, Germany).
10
RNA-seq library prep from CoIP samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA samples from CoIP_1 (150 ng), CoIP_2 (300 ng) and CoIP_3 (225 ng) samples were subjected to alkaline hydrolysis for 5 min at 96°C. RNA was precipitated and end-repaired before being converted to library using NEBNext©Small RNA Library kit (NEB, USA). Library quality and quantity were assessed using a High Sensitivity DNA chip on a Bioanalyzer 2100 and using Qubit 2.0 fluorimeter, respectively. Libraries were multiplexed and subjected for high-throughput sequencing using an Illumina HiSeq 1000 instrument with a 50 bp single-end read mode.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!