Truseq small rna library

Manufactured by Illumina

Sourced in United Kingdom

The TruSeq Small RNA Library Prep Kit is a tool designed for preparing small RNA samples for sequencing on Illumina platforms. It enables the generation of stranded small RNA libraries from total RNA samples, capturing a wide range of small RNA species including microRNAs, siRNAs, and piRNAs.

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9 protocols using truseq small rna library

Illumina RNA Sequencing Protocol

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Total RNA was sequenced on an Illumina HiSeq 2500 sequencing system using a TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero treatment (Illumina) or the TruSeq Small RNA Library (Illumina) at the Institute of Applied Genomics (Udine, Italy). An average of about 21 million 125 base pairs long paired-end reads or 17 million 50 base pairs long single-end reads were produced for each sample for RNA-seq or small RNA-seq, respectively. Bioinformatics analysis is described in detail in the Supplemental Experimental Procedures.
RNA-seq raw data have been deposited at the GEO (GEO: GSE94888).

De Santis R., Santini L., Colantoni A., Peruzzi G., de Turris V., Alfano V., Bozzoni I, & Rosa A. (2017). FUS Mutant Human Motoneurons Display Altered Transcriptome and microRNA Pathways with Implications for ALS Pathogenesis. Stem Cell Reports, 9(5), 1450-1462.

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RNA Isolation and Transcriptome Analysis

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RNA was extracted from cells using the miRNeasy miRNA isolation mini kit (#217004, Qiagen). To quantify mRNA of interest, cDNA synthesis and quantitative PCR (qPCR) were conducted using the GoTaq 2-Step RT-qPCR dye-based detection system (#A6010, Promega) and a Roche 480 Lifecycler (Roche, Sussex, UK). Quantification of microRNAs was achieved using the MystiCq microRNA cDNA synthesis mix and MystiCq microRNA qPCR dye-based assay primer system (#MIRRT, Sigma). Primers are detailed in Supplementary Table 3. For RNAseq samples were prepared using the Truseq small RNA library (#RS-200-0012, Illumina, Essex, UK), Truseq Stranded RNA library (#20020597, Illumina) and sequenced using the NextSeq 500 High Output Run (150 cycles) for two biological replicates. Two biological replicates were sequenced and analysis conducted using Cutadtapt, SHRiMP, SAMtools for trimming, mapping to GRch38 build, generating the raw counts. Data available from the Gene Expression Omnibus GSE117744. FunRich software was used to conduct enrichment analysis with Fishers exact test to generate P values [45 (link)].

Probert C., Dottorini T., Speakman A., Hunt S., Nafee T., Fazeli A., Wood S., Brown J.E, & James V. (2018). Communication of prostate cancer cells with bone cells via extracellular vesicle RNA; a potential mechanism of metastasis. Oncogene, 38(10), 1751-1763.

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Small RNA Sequencing Library Preparation

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Adopting the Illumina TruSeq Small RNA library protocol outlined in TruSeq Small RNA Sample Preparation Guide, the precursors RNA sequencing library was constructed. To 1 μg of total RNA 3′ adaptors were ligated followed by 5′ adaptor ligation. Reverse transcription of the ligated products was carried out by Superscript III Reverse transcriptase (Invitrogen, 18064014) after priming with reverse transcription primers. cDNA enrichment and barcoding by PCR (15 cycles, 98 °C for 10 s, 60 °C for 30 s and 72 °C for 15 s) were subsequently performed and products were cleaned by polyacrylamide gel. Libraries in the range of 160–300 bp (including adaptors) were size selected and gel eluted. Salt precipitation, quantity and quality checking were performed as mentioned in sRNA library preparation. Illumina sequencing (Illumina Next Seq 500, Genotypic Pvt. Ltd., Bangalore, India) was performed for two libraries in paired ends to cover the 160–300 nt sequences.

Velayudha Vimala Kumar K., Srikakulam N., Padmanabhan P, & Pandi G. (2017). Deciphering microRNAs and Their Associated Hairpin Precursors in a Non-Model Plant, Abelmoschus esculentus. Non-Coding RNA, 3(2), 19.

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Small RNA Sequencing Protocol

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Extracted samples were shipped to Macrogen Inc (Seoul, South Korea) for small RNA sequencing. Illumina TruSeq Small RNA Library construction of the 15 samples was completed. Sequencing was done on an Illumina HiSeq 2500 machine with 8 million reads per sample.

DeRaedt S., Bierman A., van Heusden P., Richards C, & Christoffels A. (2022). microRNA profile of Hermetia illucens (black soldier fly) and its implications on mass rearing. PLoS ONE, 17(3), e0265492.

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Comprehensive Multiomics Analysis of Gene Expression

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RNA was extracted from cells using the miRNeasy miRNA isolation mini kit (#217004, Qiagen). To quantify mRNA of interest, cDNA synthesis and quantitative PCR (qPCR) were conducted using the GoTaq 2-Step RT-qPCR dye based detection system (#A6010, Promega) and a Roche 480 Lifecycler (Roche, Sussex, UK). Quantification of microRNAs was achieved using the MystiCq microRNA cDNA synthesis mix and MystiCq microRNA qPCR dye based assay primer system (#MIRRT, Sigma). Primers are detailed in Supplementary Table 3. For RNAseq samples were prepared using the Truseq small RNA library (#RS-200-0012, Illumina, Essex, UK), Truseq Stranded RNA library (#20020597, Illumina) and sequenced using the NextSeq 500 High Output Run (150 cycles) for two biological replicates. Two biological replicates were sequenced and analysis conducted using Cutadtapt, SHRiMP, SAMtools for trimming, mapping to GRch38 build, generating the raw counts. Data available from the Gene Expression Omnibus (GEO) GSE117744. FunRich software was used to conduct enrichment analysis with Fishers exact test to generate P values (45 ).

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Small RNA Sequencing of Schistosoma haematobium

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Methods used for the isolation and quality assessment of total RNA were described previously [5 (link)]. The sRNA libraries representing male or female adults of S. haematobium (accession numbers: SRR6655496 and SRR6655494) were constructed (TruSeq Small RNA Library, Illumina) and sequenced (HiSeq 2500 sequencing platform, Illumina) according to manufacturer’s instructions. Reads were filtered for low quality (Phred score: < 35) and adapters removed using Trimmomatic [29 (link)], retaining only reads with the 3’-adapter plus ≥ 8 bases.

Stroehlein A.J., Young N.D., Korhonen P.K., Hall R.S., Jex A.R., Webster B.L., Rollinson D., Brindley P.J, & Gasser R.B. (2018). The small RNA complement of adult Schistosoma haematobium. PLoS Neglected Tropical Diseases, 12(5), e0006535.

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Single-Cell RNA Sequencing of Triptolide-Treated Drosophila

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For each condition 20 10^ˆ6 of Triptolide treated Drosophila cells were spiked with 2 10^ˆ6 untreated mouse embryonic stem cells. scRNA protocol was adapted from (Nechaev et al., 2010 (link)). Cells were re-suspended in ice-cold lysis buffer (10mM Tris (pH = 7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA, 0.5% NP40), incubated 10min on ice, span down. Nuclei were washed with ice cold (10mM Tris (pH = 7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA) and nuclear pellets were dissolved in Trizol (Thermofisher). RNA was size selected (17-200bp) using a two-step column purification strategy (RNA clean and concentrator, Zymo-R1016). 10 μg of purified RNA was successively treated by 5′ dephosphorylation - 20U at 37°C for 30min (Epicenter - RP8092H); 5′ terminator exonuclease - 1U in Buffer A at 30°C for 60min (Epicenter - TER51020); cap-clip decapping enzyme – 5U at 37°C for 90min. After each reaction, short RNA was column purified (RNA clean and concentrator, Zymo-R1016). The resulting RNA was used for library preparation using TruSeq small RNA library (Illumina). Libraries were purified on 6% TBE gels (150-300bp – Novex - EC6265BOX). Size distribution of the libraries were controlled on Bioanalyser High sensitivity (Agilent 5067-4626). Two biologically independent inhibition time courses were performed. The samples were run on an Illumina NextSeq generating 38bp paired-end reads.

Krebs A.R., Imanci D., Hoerner L., Gaidatzis D., Burger L, & Schübeler D. (2017). Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters. Molecular Cell, 67(3), 411-422.e4.

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miRNA Sequencing Protocol for MCF-7 Cells

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The total RNA from both MCF-7 cells treated with TChal and MCF-7 control treated with DMSO were extracted using the miRNeasy Isolation (Qiagen, Germantown, MD, USA) and the preparation of the miRNA library used the TruSeq Small RNA Library (Illumina, San Diego, CA, USA) kit, and both assays were done following the manufacturer’s instructions. The integrity of each miRNA library was analyzed by a bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) as per the manufacturer’s instructions. For the library construction, the total RNA sample was used to the manufacturer’s instructions, and adaptors were added to the small RNAs. The small RNAs were converted into cDNAs by PCR, then to obtain the mature miRNA, electrophoresis was performed using the polyacrylamide gel (12%), and the mature miRNAs were between 145–160 pb, so the fragments were cut off the gel. Afterward, the fragments were purified, and the cDNA library was quantified by KAPA library quantification (Illumina sequencing platforms). Lastly, the samples were sequenced using a MiSeq v2 Reagent kit (Illumina, San Diego, CA, USA) on a Mi-seq system.

Komoto T.T., Nishimura F.G., Evangelista A.F., de Freitas A.J., da Silva G., Silva W.A., Peronni K., Marques M.M., Marins M, & Fachin A.L. (2023). Exploring the Therapeutic Potential of trans-Chalcone: Modulation of MicroRNAs Linked to Breast Cancer Progression in MCF-7 Cells. International Journal of Molecular Sciences, 24(13), 10785.

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Inducing Asexual Male Production

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We obtained male RNA samples from line 409, which produces wingless males [25 (link)]. We induced asexual females to produce males by moving them from a summer to a fall photoperiod. Once an asexual female begins producing males (~three generations later), they continue to produce males until death. We identified females producing males by isolating them onto individual, small Petri dishes and following their offspring until adulthood. Male-producing females were then allowed to lay offspring for several days so that we could collect first instar nymphs (one-day-old) and second instar nymphs (three days old). We also dissected the females to collect embryos. We, therefore, collected samples from three developmental stages: embryos, first instar nymphs, and second instar nymphs. Each sample was a pool of 15–20 individuals. We extracted total RNA separately from each pool using a standard TRizol protocol plus Dnase1 treatment and then pooled them in equal proportions in order to sequence a single sample. Small RNAs were size selected, processed into an Illumina TruSeq Small RNA library, and sequenced using Illumina single-end, 50-nt reads in a single lane by the University of Rochester’s Genomic Center. Small RNA data are available in the NCBI BioProject PRJNA732656.

Liu X., Culbert E.L, & Brisson J.A. (2021). Male-Biased microRNA Discovery in the Pea Aphid. Insects, 12(6), 533.

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