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Multiplex small rna library prep set

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The Multiplex Small RNA Library Prep Set is a laboratory equipment product designed for the preparation of small RNA libraries. It enables the simultaneous construction of multiple small RNA libraries from various samples, facilitating high-throughput analysis. The core function of this set is to provide a standardized and efficient workflow for the preparation of small RNA libraries.

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Lab products found in correlation

Novaseq 6000 system, by Illumina (2 mentions) Bioanalyzer 2100 system, by Agilent Technologies (2 mentions) Hiseq 2500, by Illumina (1 mentions) Longamp taq 2 master mix, by New England Biolabs (1 mentions) Hiseq 2500 platform, by Illumina (1 mentions) Artseq, by Illumina (1 mentions) Artseq ribosome profiling kit, by Illumina (1 mentions) Hiseq sr cluster kit v4 cbot, by Illumina (1 mentions) Hiseq v4 single read flow cell, by Illumina (1 mentions) Hiseq sbs kit v4 sequencing reagents, by Illumina (1 mentions)

5 protocols using multiplex small rna library prep set

1

Characterizing BLV miRNA Expression Using RNA-seq

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HEK293T cells (6-well format) were transfected with 2 µg of BLV miRNA expression vectors using Lipofectamine 2000. RNA was extracted 48 h post transfection and fractionated on a 15% PAGE-urea gel. Small RNAs (<70 nt) were isolated as previously described (31 (link)) and treated with or without RNA 5′ polyphosphatase. RNA was then recovered via ammonium acetate precipitation. 5′-end characterization as described above, was performed to confirm miRNA expression and dephosphorylation by the RNA 5′ polyphosphatase treatment. Small RNA libraries were then prepared (GSAF, UT Austin, USA) for Illumina small RNA sequencing (RNA-seq) using the multiplex small RNA library prep set (New England Bio Labs) and sequenced on a Illumina HiSeq 2500. Adapter sequences were trimmed from the reads using custom Python scripts. The preprocessed reads were then mapped to the respective pri-miRNA sequences using the SHRiMP2 software package (33 (link)). To analyze the fold change of the 5′-start position of putative B5 pre-miRNAs with RNA 5′ polyphosphatase, we counted RNA-seq reads of RNAs approximately pre-miRNA length (>50 nt), in which the 5′-nucleotide perfectly mapped to the BLV B5 miRNA locus (30 nt upstream and 54 nt downstream of the 5′-end of BLV-miR-B5 5p).
Burke J.M., Bass C.R., Kincaid R.P, & Sullivan C.S. (2014). Identification of tri-phosphatase activity in the biogenesis of retroviral microRNAs and RNAP III-generated shRNAs. Nucleic Acids Research, 42(22), 13949-13962.
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2

Small RNA Sequencing of Extracellular Vesicles

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Total RNA of M2-EVs was extracted using TRIzol and then treated with DNase I to deplete the genomic DNA. The Multiplex Small RNA Library Prep Set (New England Biolabs) was used to produce small RNA libraries. cDNAs were prepared using adaptor-specific primers, and DNA fragments (∼140–160 bp) were recovered. The library quality was evaluated using DNA High Sensitivity Chips on an Agilent Bioanalyzer 2100 system. Sequencing of libraries was conducted on an Illumina Novaseq 6000 system and 50 bp single-end reads were generated. All identical sequences with sizes of 18 to 32 nt were counted and removed from the initial data set. After elimination of nonmiRNAs (rRNA, tRNA, snoRNA, etc.), the expression of miRNAs was analyzed using the perfectly matched sequences from the BLAST search of miRbase (version 21.0). The cluster plot of miRNAs was generated by an online platform (https://www.omicsolution.org/wkomics/main/). The miRNA-gene interaction was analyzed using miRNet (https://www.mirnet.ca/) [20 (link)].
Wang Y., Liu S., Li L., Li L., Zhou X., Wan M., Lou P., Zhao M., Lv K., Yuan Y., Chen Y., Lu Y., Cheng J, & Liu J. (2022). Peritoneal M2 macrophage-derived extracellular vesicles as natural multitarget nanotherapeutics to attenuate cytokine storms after severe infections. Journal of Controlled Release, 349, 118-132.
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3

Small RNA Sequencing from Fecal Samples

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Fecal samples were used for total RNA extraction using a miRNA isolation kit with phenol (Cat#AM1561, Invitrogen). Multiplex Small RNA Library Prep Set (NEBNext) were used to construct the small RNA-seq libraries. PCR amplification were performed using LongAmp Taq 2× Master Mix (Cat#M0287L, NEB) and quality was determined on an Agilent Bioanalyzer 2100 system. Then, the library preparations were constructed on an Illumina Hiseq 2500 platform, All the 18 ~ 26 nucleotide-length unique sequences were mapped to specific species precursors in miRBase 22.0. Different miRNA expression was analyzed using Student t test.
Zhou X., Liu Y., Xiong X., Chen J., Tang W., He L., Zhang Z., Yin Y, & Li F. (2022). Intestinal accumulation of microbiota-produced succinate caused by loss of microRNAs leads to diarrhea in weanling piglets. Gut Microbes, 14(1), 2091369.
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4

Ribosome Profiling of Platelets

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For ribosome footprint profiling experiments, 1 × 109 platelets were treated with cycloheximide (100 mg/ml) for 1 min at RT to preserve ribosomes as natively attached to mRNAs.35 (link) The platelets were lysed in Mammalian Lysis Buffer (ARTseq, Epicentre). Total RNA and ribosome-protected read RNAs (RPRs) were prepared per the manufacturer’s instructions (ARTseq-R ibosome Profiling Kit, Epicentre). Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single-read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (GD- 401– 4001). Following transfer of the flow cell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50-cycle single-read sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC- 401– 4002). Ribosome profiling (eukaryote) library preparation was completed using the NEBNext Multiplex Small RNA Library Prep Set.
Eustes A.S., Campbell R.A., Middleton E.A., Tolley N.D., Manne B.K., Montenont E., Rowley J.W., Krauel K., Blair A., Guo L., Kosaka Y., Medeiros- de- Moraes I.M., Lacerda M., Hottz E.D., Neto H.C., Zimmerman G.A., Weyrich A.S., Petrey A, & Rondina M.T. (2021). Heparanase expression and activity are increased in platelets during clinical sepsis. Journal of thrombosis and haemostasis : JTH, 19(5), 1319-1330.
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5

Small RNA Sequencing of M2-EVs

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Total RNA of M2-EVs was extracted using Trizol and then treated with DNase I to deplete the genomic DNA. The Multiplex Small RNA Library Prep Set (New England Biolabs) was used to produce small RNA libraries. cDNAs were prepared using adaptor-specific primers, and DNA fragments (∼140-160 bp) were recovered.
The library quality was evaluated using DNA High Sensitivity Chips on an Agilent (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted March 14, 2022. ; https://doi.org/10.1101/2022.03.13.484180 doi: bioRxiv preprint Bioanalyzer 2100 system. Sequencing of libraries was conducted on an Illumina Novaseq 6000 system and 50 bp single-end reads were generated. All identical sequences with sizes of 18 to 32 nt were counted and removed from the initial data set.
After elimination of non-miRNAs (rRNA, tRNA, snoRNA, etc.), the expression of miRNAs was analyzed using the perfectly matched sequences from the BLAST search of the miRbase (version 21.0). The cluster plot of miRNAs was generated by an online platform (https://www.omicsolution.org/wkomics/main/). The miRNA-gene interaction was analyzed using miRNet (https://www.mirnet.ca/) [22] .
Wang Y., Liu S., Li L., Li L., Zhou X., Wan M., Lou P., Zhao M., Lv K., Yuan Y., Chen Y., Lu Y., Cheng J., & Liu J. (2022). Peritoneal M2 macrophage-derived extracellular vesicles as natural multi-target nanotherapeutics to attenuate cytokine storm after severe infections.
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