Trizol (Invitrogen, USA) was applied to extract total RNA from SKOV-3 cells. Hieff NGSTM MaxUp Dual-mode mRNA Library Prep Kit for Illumina® and NEBNext UltraTM small RNA Sample Library Prep Kit for Illumina (NEB, USA) were used to construct RNA-seq libraries. Samples were sequenced by Illumina (Illumina, USA) after library preparation. mRNA and miRNA sequencing reads were aligned using the HISAT2 and Bowtie aligner, respectively. The reads data were mapped onto the human reference genome (hg19). mRNA and miRNA transcript quantification were performed using miRDeep2 and StringTie, respectively, furthermore, differential expression analysis was performed by DESeq2.
Nebnext ultra small rna sample library prep kit
Manufactured by Illumina
Sourced in United States, China
The NEBNext Ultra small RNA Sample Library Prep Kit is a laboratory equipment product designed for preparing small RNA samples for sequencing. It provides the necessary reagents and protocols to extract, purify, and prepare small RNA samples for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Qubit 2, by Agilent Technologies (3 mentions)
Hiseq 2500 platform, by Illumina (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
Hieff ngstm maxup dual mode mrna library prep kit, by Illumina (1 mentions)
Quick rna tissue insect microprep kit, by Zymo Research (1 mentions)
Hiseq x ten, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
Feasible barcode primers, by Illumina (1 mentions)
Hiseq4000 analyzer, by Illumina (1 mentions)
8 protocols using nebnext ultra small rna sample library prep kit
1
Transcriptome Analysis of SKOV-3 Cells
2
Identifying Small RNA Libraries from Diamondback Moth
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To build two small RNA (sRNA) libraries of the GC strain, for each library, the hemolymph of 30 first-day 4th instar larvae were dissected and pooled. Hemolymph samples were collected in the lysis buffer of a Quick-RNA Tissue/Insect Microprep kit (#R2030, ZymoResearch, Irvine, CA, USA) using a glass needle, and centrifuged at 13,000× g for 1 min at room temperature for RNA extraction, using the same kit according to the manufacturer’s instructions. Sequencing libraries were constructed using the NEBNext® UltraTM small RNA Sample Library Prep Kit for Illumina, and the sequencing procedure was performed by Biomarker Technologies Corporation (Illumina HiSeq2500, Beijing, China). Clean reads were compared to the known plant microRNA (miRNA) precursors deposited in the online miRBase (version 21, http://www.mirbase.org/ ). Unknown plant miRNAs were inferred by miRdeep2 software using the settings of “-g -1 -l 250 -b 0” based on the information of precursors in the genome location and their structural energies [34 (link)]. The identified miRNAs that were supposed to be plant-derived were further used to map the P. xylostella genome using Bowtie [35 (link)] with a default parameter to exclude the possibility of the presence of these miRNAs.
3
RNA Sequencing Library Preparation
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RNA samples undergo a series of strict quality control. Qualified samples are used for library construction. Library construction is strictly in accordance with NEBNext Ultra Small RNA Sample Library Prep Kit for Illumina, qualified libraries for high-throughput sequencing. The sequencing platform was Illumina HiSeq X Ten, and read length was single-end (SE) 50 nt.
4
miRNA Sequencing and Analysis of EVs
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For miRNA sequencing, RNA was isolated by Trizol (Life Technologies) extraction from EVReN and EV293. Qubit 2.0 and Agilent 2100 bioanalyzer were used to quantify the samples. The cDNA libraries were produced using a NEBNext Ultra small RNA Sample Library Prep Kit for Illumina according to the manufacturer's instructions. Subsequently, the library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated. Using Bowtie software, the clean reads were compared with Silva database, GtRNAdb database, Rfam database and Repbase database sequence alignment to filter ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and other ncRNA and repeats. The remaining reads were used to detect known miRNA and new miRNA predicted by comparing with known miRNAs from miRBase. The miRNA levels were calculated and normalized to transcripts per million (TPM). A heatmap analysis of miRNA expression levels was created based on the TPM values of miRNAs in EVReN and EV293 (using TPMaverage > 1000, 1.5-fold change and p < 0.05 as the threshold cutoff). Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to determine enriched pathways involved in the predicted target genes of differentially expressed miRNAs.
5
RNA Extraction and Sequencing of Jejunum Samples
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The sectioned jejunum samples were flash frozen in liquid nitrogen and stored at -80°C until RNA processing. Total RNA was extracted from the inoculated and control tissues using the Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. The cDNA library was established according to the protocol of the NEB Next Ultra Small RNA Sample Library Prep Kit by Illumina. Briefly, 3’ and 5’ adaptor ligation was followed by first-strand cDNA synthesis. PCR enrichment was followed by clean up and size selection. After library establishment, the concentration of the library was measured using a Qubit 2.0 (Life Technologies). The concentration of the library was diluted to 1 ng/μl. The insert size was measured with an Agilent 2100 bioanalyzer [24 (link)]. To ensure quality, the q-PCR method was used to quantify the concentration of the library. Finally, Illumina HiSeq2500 was used for high-throughput sequencing, and the sequencing read length was set to single-end (SE) 50 nt.
6
Small RNA Sequencing Library Preparation
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Small RNA libraries were constructed using a NEB Next Ultra Small RNA Sample Library Prep Kit for Illumina according to the manufacturer’s instructions. Reverse transcription primer was hybridized after 3′ adaptor ligation of 10 ng RNA per sample, following 5′ adaptor ligation. Twelve cycles of PCR were performed using Illumina feasible barcode primers after first-strand cDNA synthesis. The prepared libraries were resolved on a native 7% polyacrylamide gel. DNA fragments corresponding to 160–180 bp (including 3′ and 5′ adaptors) were recovered in 10 μL of DNase- and RNase-free water.
Libraries were quantified by the Agilent 2100 bioanalyzer using DNA 1000 chips. A total of 36 sequencing libraries were pooled into a single sequencing lane and sequenced using an Illumina HiSeq4000 analyzer (Illumina, USA).The miRNA sequencing data were quantified, and sequences with a length of more than 18 nt were aligned against miRBase (release 21). The miRNA profiling was normalized using reads per million (RPM) mappable miRNA sequences. Analysis of the differentially expressed miRNAs between the two groups was performed using edgeR software. The Gene Expression Omnibus (GEO) database accession number for the miRNA profile data reported in this study is GEO:GSE147517 .
Libraries were quantified by the Agilent 2100 bioanalyzer using DNA 1000 chips. A total of 36 sequencing libraries were pooled into a single sequencing lane and sequenced using an Illumina HiSeq4000 analyzer (Illumina, USA).The miRNA sequencing data were quantified, and sequences with a length of more than 18 nt were aligned against miRBase (release 21). The miRNA profiling was normalized using reads per million (RPM) mappable miRNA sequences. Analysis of the differentially expressed miRNAs between the two groups was performed using edgeR software. The Gene Expression Omnibus (GEO) database accession number for the miRNA profile data reported in this study is GEO:
7
miRNA Sequencing and Expression Analysis
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For miRNA sequencing, RNA was isolated by Trizol (Life Technologies) extraction from ReN-NV and 293-NV. Qubit 2.0 and Agilent 2100 bioanalyzer were used to quantify the samples. The cDNA libraries were produced using a NEBNext Ultra small RNA Sample Library Prep Kit for Illumina according to the manufacturer’s instructions. Subsequently, the library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated. Using Bowtie software, the clean reads were compared with Silva database, GtRNAdb database, Rfam database and Repbase database sequence alignment to filter ribosomal RNA(rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA)and other ncRNA and repeats. The remaining reads were used to detect known miRNA and new miRNA predicted by comparing with known miRNAs from miRBase. The miRNA levels were calculated and normalized to transcripts per million (TPM). A heatmap analysis of miRNA expression levels was created based on the TPM values of miRNAs in ReN-NV and 293-NV (using TPMaverage > 1000, 1.5-fold change and P < 0.05 as the threshold cutoff).
8
Small RNA-Seq Protocol for miRNA Profiling
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For sequencing, RNA was isolated by Trizol (Life Technologies) extraction from ReN-NV and 293-NV. Qubit 2.0 and Agilent 2100 bioanalyzer were used to quantify the samples. The cDNA libraries were produced using a NEBNext Ultra small RNA Sample Library Prep Kit for Illumina according to the manufacturer's instructions. Subsequently, the library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated. Using Bowtie software, the clean reads were compared with Silva database, GtRNAdb database, Rfam database and Repbase database sequence alignment to lter ribosomal RNA(rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA)and other ncRNA and repeats. The remaining reads were used to detect known miRNA and new miRNA predicted by comparing with known miRNAs from miRBase. The miRNA levels were calculated and normalized to transcripts per million (TPM).A heatmap analysis of miRNA expression levels was created based on the TPM values of miRNAs in ReN-NV and 293-NV (using TPM average > 1000, 1.5-fold change and p < 0.05 as the threshold cutoff ).
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