Nebnext small rna library prep kit

Manufactured by Illumina

The NEBNext Small RNA Library Prep Kit is a laboratory equipment product designed for the preparation of small RNA libraries. It provides a standardized workflow for the generation of sequencing-ready libraries from small RNA samples.

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10 protocols using nebnext small rna library prep kit

Small RNA Sequencing Library Preparation

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Trizol (Invitrogen) was used to extract RNA from cells, after which an Agilent 2200 machine was used to assess RNA quality prior to storage at −80°C. RNA samples with an integrity score >7.0 were used to prepare cDNA libraries.
To produce small RNA sequencing libraries, we utilized an NEBNext Small RNA Library Prep Kit for Illumina. Briefly, RNA was ligated to the provided 5′ and 3′ adapters, followed by first strand cDNA synthesis. Index PCR was then used in order to apply index sequences and Illumina sequence adapters. Library purification was then performed, and a Bioanalyzer 2200 (Agilent, CA, USA) was used for quality control followed by sequencing on a HiSeq X‐ten platform (Illumina, CA, USA) using 150 bp paired‐end reads.

Wang T., Cao L., He S., Long K., Wang X., Yu H., Ma B., Xu X, & Li W. (2020). Small RNA sequencing reveals a novel tsRNA‐06018 playing an important role during adipogenic differentiation of hMSCs. Journal of Cellular and Molecular Medicine, 24(21), 12736-12749.

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Cross-Linking Immunoprecipitation (CLIP) Protocol

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CLIP was performed as previously reported^{18 (link)} with some modifications. Briefly, the whole cell lysate from cross-linked (twice by 150 mJ per cm² of 365 nm UV light) PANC-1 cells were isolated and sonicated, followed by treatment with DNase I (0.5 U/μl, 37 °C for 5 min) and RNase TI (0.2 U/μl, 22 °C for 15 min). Pre-washed Dynabeads protein A/G (Millipore) conjugated with 10 μg antibodies against CSTF2, METTL3, or IGF2BP2 were then incubated with the extraction at 4 °C overnight with rotating. After substantial washing of beads, end repair was performed by using T4 PNK (NEB). RNA was then treated with proteinase K (37 °C for 30 min), acidic phenol/chloroform extraction, and ethanol precipitation, and was subsequently used for library construction by using NEBNext small RNA library prep kit (E7330S) and sequenced on Illumina Hiseq4000. For CLIP-qPCR, the input and immunoprecipitated RNA samples were recovered as described above. cDNA was synthesized with SuperScript III RT (Invitrogen) and random hexamer primers (Invitrogen) and subject to qRT-PCR using specific primers shown in Supplementary Table 3.

Zheng Y., Li X., Deng S., Zhao H., Ye Y., Zhang S., Huang X., Bai R., Zhuang L., Zhou Q., Li M., Su J., Li R., Bao X., Zeng L., Chen R., Zheng J., Lin D., He C., Zhang J, & Zuo Z. (2023). CSTF2 mediated mRNA N6-methyladenosine modification drives pancreatic ductal adenocarcinoma m6A subtypes. Nature Communications, 14, 6334.

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Small RNA Sequencing for AML Analysis

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Small RNA species were sequenced using total RNA from the primary AML samples and normal donor samples using the NEBNext Small RNA Library Prep Kit for Illumina following the recommended protocol. Briefly, 100 ng of DNAase-treated input RNA was used for 3’ ligation, RT primer hybridization, reverse-transcriptase, and 5’ ligation reactions. Library molecules were then amplified with sample-specific indexed adapters for 12–15 cycles, and then size selected via electrophoresis on a bluepippin instrument to obtain molecules with library inserts between 17 and 200 nucleotides. Libraries were then multiplex sequenced on a MiSeq instrument, and sequencing reads were mapped with bwa mem (Li, 2016 ) using the parameters ‘-k 15 -T 17’ to yield alignments for short library inserts. Read counts for annotated genes in the gencode version 19 database were then generated using ‘featureCounts’ (Liao et al., 2014 (link)), with the parameters ‘-s 1 -t exon -g transcript_id -Q 1 -O’ to accurately count reads in small RNA annotations that are occasionally embedded in larger gene annotations. Normalized counts and differentially expressed species were obtained with DESeq2 (Love et al., 2014 (link)).

Spencer D.H., Russler-Germain D.A., Ketkar-Kulkarni S., Helton N.M., Lamprecht T.L., Fulton R.S., Fronick C.C., O’Laughlin M., Heath S.E., Shinawi M., Westervelt P., Payton J.E., Wartman L.D., Welch J.S., Wilson R.K., Walter M.J., Link D.C., DiPersio J.F, & Ley T.J. (2017). CpG island hypermethylation mediated by DNMT3A is a consequence of AML progression. Cell, 168(5), 801-816.e13.

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Small RNA Library Preparation for Illumina Sequencing

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The NEBNext Small RNA Library Prep kit (NEB) was used to generate Illumina sequencing libraries. The libraries were amplified through 15 cycles of PCR. For the E. coli libraries sequencing was performed on an Illumina MiSeq Instrument with single reads of 100 bases using V3 reagent kit. For the mouse microbiome, the libraries were sequenced on an Illumina GAII platform. All the raw reads have been deposited in the European Nucleotide Archive (ENA) website under the accession number PRJEB9717, (http://www.ebi.ac.uk/ena/data/view/PRJEB9717).

Ettwiller L., Buswell J., Yigit E, & Schildkraut I. (2016). A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics, 17, 199.

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Illumina RNA-Seq Library Prep

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Purified exRNA was made into Illumina Tru-Seq RNA-Sequencing libraries, and samples were indexed using the NEBNext Small RNA Library prep kit (Illumina, San Diego, CA). Libraries were amplified for 12–18 cycles according to the manufacturer’s instructions. Amplified libraries were sized-fractionated using Sage Bioscience Pippin prep station on 2% agarose gels (Sage Bio cat # SDF2010) with a collection range of 135 bp—200 bp. The size and concentration of the purified libraries were analyzed using an Agilent 2100 Bioanalyzer high sensitivity DNA chip. Libraries were pooled in equal molar amounts for multiplex sequencing on an Illumina MiSeq sequencer.

Yan I.K., Wang X., Asmann Y.W., Haga H, & Patel T. (2016). Circulating Extracellular RNA Markers of Liver Regeneration. PLoS ONE, 11(7), e0155888.

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Small RNA Profiling of Wilms Tumors

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Small RNA libraries were prepared from 32 Wilms tumor samples with adequate RNA. For small RNA, libraries were prepared and sequenced at DNALink, Inc. using the NEBNext Small RNA Library Prep Kit for Illumina, and reads were sequenced on the Illumina NextSeq 500 platform. Trimmed reads were mapped to miRbase using miRDeep2 and normalized to the spike-in control. Differential expression analysis was performed using DESeq2.

Xu L., Desai K., Kim J., Zhou Q., Guo L., Xiao X., Zhang Y., Zhou L., Yuksel A., Catchpoole D.R., Amatruda J.F, & Chen K.S. (2023). WILMS TUMOR MUTATIONAL SUBCLASSES CONVERGE TO DRIVE CCND2 OVEREXPRESSION. medRxiv.

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RNA-Seq of Frozen Liver Samples

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Livers were frozen and stored in RNA‐later. Total RNA was extracted using TRIzol (Life Technologies). Samples were processed at Mayo Clinic Medical Genome Facility Sequencing Core. Illumina Tru‐Seq RNA‐sequencing (RNA‐seq) libraries were prepared, and samples were indexed using the NEBNext Small RNA Library prep kit for Illumina (San Diego, CA). Libraries were amplified 12 to 18 cycles. Amplified libraries were size selected using the Pippin prep (Sage Bioscience) on 2% agarose gels with a size range from 135 to 200 base pairs (bp). Quality control analysis for size and concentration of purified libraries was performed using an Agilent 2100 bioanalyzer high‐sensitivity DNA chip. Libraries were pooled in equal molar amounts for multiplex sequencing on an Illumina HiSeq 2000 sequencer for paired ends.

Matsuda A., Ishiguro K., Yan I.K, & Patel T. (2019). Extracellular Vesicle‐Based Therapeutic Targeting of β‐Catenin to Modulate Anticancer Immune Responses in Hepatocellular Cancer. Hepatology Communications, 3(4), 525-541.

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Small RNA Sequencing Library Preparation

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Spike-in RNAs (Exiqon) were added into 1 μg total RNA before library construction. Small RNA sequencing libraries were generated using NEBNext Small RNA library Prep kit and NEBNext multiplex oligos for Illumina according to the manufacturer's instructions (NEB). The final libraries were purified from 6% PAGE gel, and their concentrations were measured using Qubit fluorometric assay (Life Technologies).

Cass A.A., Bahn J.H., Lee J.H., Greer C., Lin X., Kim Y., Hsiao Y.H, & Xiao X. (2016). Global analyses of endonucleolytic cleavage in mammals reveal expanded repertoires of cleavage-inducing small RNAs and their targets. Nucleic Acids Research, 44(7), 3253-3263.

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RNA-seq and csRNA-seq library preparation

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For RNA-seq, strand-specific, paired-end libraries were prepared from total RNA by ribosomal depletion using the yeast Ribo-Zero rRNA removal kit (Illumina) and then using the TruSeq stranded total RNA-seq kit (Illumina) according to the manufacturer’s instructions. Next, 100 bases were sequenced from both ends using a NovaSeq 6000 system according to the manufacturer’s instructions (Illumina).
csRNA-seq was performed as described previously (15 (link)). Small RNAs (sRNAs) of ∼20 to 60 nucleotides (nt) were size selected from 0.4 to 2 μg of total RNA by denaturing gel electrophoresis. A 10% input sample was taken aside, and the remainder was enriched for 5′-capped RNAs. Monophosphorylated RNAs were selectively degraded by Terminator 5′-phosphate-dependent exonuclease (Lucigen), and RNAs were 5′ dephosphorylated by quickCIP (New England BioLabs [NEB]). Input (sRNA) and csRNA-seq libraries were prepared as described previously (22 (link)) using RppH (NEB) and the NEBNext small RNA library prep kit, amplified for 14 cycles, and sequenced for single end 75 base pair reads (SE75) on the Illumina NextSeq 500 system.

Duttke S.H., Beyhan S., Singh R., Neal S., Viriyakosol S., Fierer J., Kirkland T.N., Stajich J.E., Benner C, & Carlin A.F. (2022). Decoding Transcription Regulatory Mechanisms Associated with Coccidioides immitis Phase Transition Using Total RNA. mSystems, 7(1), e01404-21.

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RNA-seq Library Preparation and Sequencing

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Cellular RNA was extracted using Trizol (Invitrogen), with RNA quality confirmed using an Agilent 2200 machine. Samples that had an RNA integrity (RIN) score > 7.0 were then used for cDNA library preparation with the NEBNext Small RNA Library Prep Kit for Illumina. This RNA was first ligated to provided 3′ and 5′ adapters, after which first strand cDNA synthesis was performed. Index sequences and Illumina sequence adapters were then applied through index PCR, after which the library was purified, assessed for quality control with a Bioanalyzer 2200 instrument (Agilent, CA, USA), and sequences using a HiSeq X-ten platform (Illumina, CA, USA) with 150-bp paired-end reads.

Wang T., Mei J., Li X., Xu X., Ma B, & Li W. (2020). A novel tsRNA-16902 regulating the adipogenic differentiation of human bone marrow mesenchymal stem cells. Stem Cell Research & Therapy, 11, 365.

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