Total RNA was prepared from a whole single adult of A. lumbricoides and A. suum respectively, using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Ten μg total RNA and Novex 15% TBE-Urea gel (Invitrogen) were used for small RNA isolation. The RNA fragments of 18–30 bases long were added with 5’ and 3’ adaptors (Illumina) to the both ends, reverse transcripted with a RT-PCR kit (Invitrogen), and sequenced employing a Solexa sequencer at Huada Genomics Institute Co. Ltd, China.
5 and 3 adaptors
Manufactured by Illumina
Sourced in China, United States
5' and 3' adaptors are laboratory equipment used in molecular biology and genomics applications. They are short DNA or RNA sequences designed to be ligated or attached to the 5' and 3' ends of nucleic acid molecules. These adaptors facilitate various downstream processing steps, such as library preparation, sequencing, and bioinformatic analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Novex 15 tbe urea gel, by Thermo Fisher Scientific (2 mentions)
Rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Mini protean tetra vertical electrophoresis system, by Bio-Rad (1 mentions)
Truseq small rna sample preparation guide, by Illumina (1 mentions)
Genome analyzer iix, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Hiseq 2000 genome analyzer, by Illumina (1 mentions)
6 protocols using 5 and 3 adaptors
1
Small RNA Sequencing of Nematodes
2
Small RNA Deep Sequencing Protocol
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Small RNA library construction and deep sequencing were performed by Welgene Biotech, Co., Ltd. (Taipei, Taiwan). Samples were prepared using an Illumina Sample Preparation kit according to the TruSeq® Small RNA Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). Subsequently, 5′ and 3′ adaptors (Illumina, Inc.) were ligated to total RNA, followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) amplification (described below). Enriched cDNA constructs were size-fractionated and purified by electrophoresis in 6% polyacrylamide gel with 30% acrylamide/Bis solution (#1610156) at a ratio of 29:1, and using a Mini-PROTEAN Tetra Vertical Electrophoresis System (Bio-Rad Laboratories, Inc.). Bands containing 18–40 nucleotide RNA fragments, 140–155 nucleotides in length with both adapters, were harvested. Libraries were sequenced using an Genome Analyzer IIx (50 cycle single read; Illumina, Inc.).
3
Small RNA Sequencing from Adult Worms
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High-quality total RNA was extracted (separately) from the entire body of a male and a female adult worm using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA yield and quality were measured spectrophotometrically (BioPhotometer, Eppendorf, Germany). The total RNA (20 μg) from each of the two samples was fractionated using Novex 6 % TBE-Urea gels (Invitrogen, Carlsbad, CA, USA), and the fragments of 18–30 nt were ligated with 5′ and 3′ adaptors (Illumina) for reverse transcription. The resultant first-strand cDNA was amplified with a small RNA primer set to enrich the libraries, and the cDNA libraries were sequenced (BGI-Shenzhen, China) using Illumina technology (HiSeq2000; sequencing length: 50 nt; paired-end).
4
Small RNA Isolation from T. gallinae
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Total RNA of T. gallinae was prepared with Trizol reagent according to the manufacturer’s protocol (Invitrogen Co. Ltd). Small RNA was prepared as described previously [20 (link)]. Briefly, small RNAs of 20–35 bases in length were isolated from 10 μg total RNA using a 15% TBE-Urea polyacrylamide gel. After adding the 5’ and 3’ adaptors (Illumina Co. Ltd), the fragments were reverse transcribed and then purified with 6% TBE PAGE gel. All gels and kits were purchased from Invitrogen Co. Ltd.
5
Small RNA Sequencing of Synthetic tasiRNA
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Small RNAs were purified from agroinfiltrated N. benthamiana leaves and subjected to Northern blot analysis as previously described [54 (link)].
RNA samples from twelve leaves (2 leaves from each plant) agroinfiltrated with two independent clones of syn-tasiR-3NCR and two independent clones of syn-tasiR-CP, either in the presence or absence of miR173 precursor, were pooled. Small RNA libraries were prepared and subjected to deep sequencing by the Beijing Genomics Institute (BGI-Shenzhen, Shenzhen, China). Briefly, between 15–30 nucleotides (nt) RNAs were purified and ligated to the Illumina 5´ and 3´ adaptors, and further converted into single-stranded cDNA and then amplified by PCR. Purified PCR products were sequenced by an Illumina HiSeq 2000 Genome Analyzer.
The high throughput sequencing data are freely available at GEO databasehttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69348 (accession number GSE69348).
RNA samples from twelve leaves (2 leaves from each plant) agroinfiltrated with two independent clones of syn-tasiR-3NCR and two independent clones of syn-tasiR-CP, either in the presence or absence of miR173 precursor, were pooled. Small RNA libraries were prepared and subjected to deep sequencing by the Beijing Genomics Institute (BGI-Shenzhen, Shenzhen, China). Briefly, between 15–30 nucleotides (nt) RNAs were purified and ligated to the Illumina 5´ and 3´ adaptors, and further converted into single-stranded cDNA and then amplified by PCR. Purified PCR products were sequenced by an Illumina HiSeq 2000 Genome Analyzer.
The high throughput sequencing data are freely available at GEO database
6
Cloning and Analysis of Small RNAs
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The cloning and analysis of small RNAs were performed according to the previous description (Ai et al., 2012; Xu et al., 2012) . A 10 µg total RNA was separated by using a Novex 15% TBE-Urea gel (Invitrogen Co. Ltd). Then, 18-30 nt RNA was purified and the 5' and 3' adaptors (Illumina) were added to each end of the fragments. After each adaptor ligation, the small RNA was purified by using 6% TBE PAGE gel. Complementary DNA (cDNA) was obtained by a reverse transcription PCR (RT-PCR) kit (Invitrogen) for reverse transcription. Finally, the RT-PCR product was purified by using a 6% TBE PAGE gel (Invitrogen) and sequenced with an Illumina-Solexa sequencer. Image and signal processing was completed using Solexa Genome Analysis System (Illumina) A file containing the trace, "base-calling" and quality score data were generated for subsequent bioinformatics analysis.
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