Total RNA was treated with RNA 5′ Polyphosphatase (Epicenter) following manufacturer's instructions, before library preparation. Libraries for sRNA sequencing were prepared using the CleanTag sRNA library prep kit according to manufacturer's instructions. Although there are other sRNA library prep methods that can reduce the quantification bias due to adapter ligation, e.g. (34 (link)), in our hands and due to the small amount of starting material, CleanTag kits yield much higher signal:background (fewer adapter–dimers) for extracellular material. For all samples, 1:2 dilutions of both adapters were used with 18 amplification cycles (TriLink BioTechnologies). Libraries of 140–170 bp in length were size-selected and sequenced on an Illumina HiSeq 2500 in high-output mode with v4 chemistry and 50 bp SE reads, by Edinburgh Genomics at the University of Edinburgh (Edinburgh, UK). This insert size was chosen to focus on the small interfering guides of exWAGO, the only Argonaute protein detected within Heligmosomoides EVs. We know that these siRNAs are mainly 23–24nt and are one of the main small RNA components of EVs (31 (link)).
18 amplification cycles
Manufactured by TriLink
The 18 amplification cycles is a function that performs a specific number of repetitions during the amplification process in a laboratory setting. It is a technical parameter that determines the number of times the target DNA sequence is replicated to generate multiple copies.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using 18 amplification cycles
1
sRNA Sequencing of Extracellular Vesicles
2
Extracellular sRNA Profiling Protocol
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Total RNA was treated with RNA 5 Polyphosphatase (Epicenter) following manufacturer's instructions, before library preparation. Libraries for sRNA sequencing were prepared using the CleanTag sRNA library prep kit according to manufacturer's instructions. Although there are other sRNA library prep methods that can reduce the quantification bias due to adapter ligation, e.g. (34) , in our hands and due to the small amount of starting material, CleanTag kits yield much higher signal:background (fewer adapterdimers) for extracellular material. For all samples, 1:2 dilutions of both adapters were used with 18 amplification cycles (TriLink BioTechnologies). Libraries of 140-170 bp in length were size-selected and sequenced on an Illumina HiSeq 2500 in high-output mode with v4 chemistry and 50 bp SE reads, by Edinburgh Genomics at the University of Edinburgh (Edinburgh, UK). This insert size was chosen to focus on the small interfering guides of exWAGO, the only Argonaute protein detected within Heligmosomoides EVs. We know that these siRNAs are mainly 23-24nt and are one of the main small RNA components of EVs (31) .
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