Modified TruSeq Small RNA Library Prep using Randomized 4N Adapters: In house 4N Protocol D

This protocol describes a library preparation method for sequencing small RNA. The method uses degenerate adapters to alleviate the problem of bias in the ligation steps during small RNA library preparation, and it optimizes several other parameters to make it appropriate for use specifically with plasma RNA


Introduction
Extracellular RNAs (exRNAs) have been identified in every biofluid that has been tested.In biofluids, they have been found in extracellular vesicles, ribonucleoprotein complexes, and lipoprotein complexes.ExRNAs are of considerable interest because they can serve as signaling molecules between cells, they have the potential to serve as biomarkers for prediction and diagnosis of disease, and exRNAs or the extracellular particles that carry them might be used for therapeutic purposes.
The Extracellular RNA Communication Consortium (ERCC) is a group of laboratories funded by the U.S. National Institutes of Health.One goal of the ERCC is to develop robust and standardized methods for collecting and processing of biofluids, separating different types of exRNA-containing particles, and isolating and analyzing exRNAs.The "Reference Profiles group":http://exrna.org/referenceprofiles/ within the consortium is tasked with collecting reliable profiles of the spectrum of extracellular RNAs found in healthy individuals.These reference profiles will serve as benchmarks for comparison with the exRNA profiles from patients with disease.The present protocol for small RNA library preparation was developed by the ERCC Reference Profiles group for use with RNA isolated from human plasma.Key elements of this small RNA library preparation method include the use of 4 random nucleotides on the end of the adapters ligated to the small RNAs of interest, the use of higher than usual adapter concentrations, and the use of increased amounts of polyethylene glycol (PEG) in the ligation steps.2][3][4] The use of random adapters is designed to alleviate that problem.The use of high adapter concentrations and macromolecular crowding agents such as PEG reduces bias by driving the ligation reactions toward completion.It should be noted that the use of improved ligation conditions and increased adapter concentrations also results in the formation of more adapter dimers, so purification of desired ligation products from such unwanted side products by size fractionation is essential in this protocol.
PCR amplification Small RNA libraries may require 10-20 cycles of PCR amplification depending on the type and amount of input.It has been shown that increased amplification does not significantly affect library bias. 1,5Increased number of PCR cycles will, however, increase the amount of adapter dimers that must be separated from the library.Typically, a single PCR and gel purification step is sufficient to remove most of the adapter dimer products from insert-containing PCR products.
Size selection Because of the large excess of adapter dimers in low-input small RNA libraries, electrophoretic purification of PCR products is often necessary.This can be done using either acrylamide or agarose gels, as long as the gel can sufficiently resolve the insert-containing fragments (== 150bp) from the adapter dim er fragm ents (= = 125bp).
A full list of the protocols developed by the ERCC is available at the "exRNA Portal":http://exrna.org/resources/protocols/, the ERCC's website.This protocol is one of four protocols relating to a 2018 Nature Biotechnology paper.
Library Preparation for small RNA sequencing using 4N adapters: In house 4N   Dilute 50% PEG to 15% PEG by adding water.

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Dry PEG until it is a pellet.

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Store in dark conditions.

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Immediately place on ice for 2 minutes.
Can either pool libraries or run libraries independently.

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Nanodrop purification -Add no more than 5 µg of PCR product, minimum in low nanograms.

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For each lane, bring total volume of library(s) + TE buffer to 30µL.