Library Preparation for small RNA sequencing using 4N adapters: In house 4N Protocol C

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. Small RNA library preparation methods that lack such degenerate adapters have been found to exhibit significant bias in the representation of different RNA sequences. [1][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. In some cases, two sequential PCR and gel purification steps might be required to remove adapter dimer products efficiently. Specific notes on these issues are included below.
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. 1,5 Increased 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. However, for low input samples, the adapters are present in such great excess over the input RNA that it can be very helpful to perform two consecutive PCR and gel purification steps. The first PCR consists of only a few cycles to prevent over-amplifying the excess of adapters. The PCR products are separated by gel electrophoresis and the eluate, now enriched for insertcontaining PCR products, is then amplified further in a second PCR using universal (not index-specific) PCR primers. A second gel purification is useful for removing residual 6 adapter contaminants.
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). Use of an automated size selection instrument like the Pippin Prep, BluePippin or PippinHT can reduce variability introduced during gel excision.
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 Protocol A Make dried PEG strip tubes. These create a high PEG concentration in a small volume for the 3' ligation.
Batches of dried PEG PCR tubes can be made as follows. Add 3 μL of 50% PEG 8000 (supplied with NEB RNA ligases) into each strip tube and dry in the speedvac using low heat (37°C). This may take 1-2 hours depending on the speedvac. Once dry, the PEG will appear as a white, flaky pellet in the bottom of the tube. Cap the tubes and store desiccated at room temperature. Note that the 50% PEG solution is very viscous, so care must be taken when pipetting. Diluting the 50% PEG to 25% and aliquoting larger volumes may improve pipetting accuracy. Positive displacement pipettes may also be helpful.

5-x μL Water 6 μL Total
The dried PEG in the strip tube will take up about 1 μL in the reaction after resuspension.

1.
Heat tube containing RNA and adapter to 70°C for 2 minutes, then snap cool on ice.
To each tube containing 7 μL of denatured RNA and adapter, add:

5' ligation
In a separate tube, add 1 μL of 5' adapter (25 μM stock concentration) per ligation and denature at 70°C for 2 minutes, then snap cool on ice.

Reverse transcription (RT)
To a new tube, add:  To purify PCR product, add 100 µL of resuspended room temperature AMPure XP beads to 13 each 50 µL PCR reaction and mix well. Allow the tube to stand at room temperature for 5 minutes and then place on magnetic stand to collect beads (approximately 2 minutes). 2) The 5' end of this adapter has an inverted dideoxy-T followed by 4 DNA bases with phosphorothioate bonds (indicated by asterisks) to prevent nuclease degradation.
3) This protocol is optimized for plasma libraries starting with ~100 μL of plasma.
Libraries with more starting material can probably tolerate higher adapter concentration and higher PEG concentration. Libraries with less starting material may need reduced adapter or PEG concentration. Caution: Differential expression analysis, i.e. comparison of RNA levels across samples, should not be done between data sets from libraries prepared using different PEG or adaptor concentrations. Relative read numbers are affected by these parameters.

4)
Incubating the reverse transcription reaction with RiboShredder RNAse blend might not be necessary.

5)
A single PCR of 15-20 cycles and gel purification may be sufficient for most plasma samples. If adapter ligation products are still a problem, the PCR and size selection can be split into 2 phases, as shown here. First, do 4 PCR cycles followed by a gel purification.
Then use the recovered DNA for a second PCR of 11-16 cycles followed by a second gel purification.
6) If an automated size selection instrument like a BluePippin or PippinHT is not available, size select a band of 149bp with any suitable gel purification system. 7) Use of a real-time master mix is not necessary, but can be helpful for determining the number of amplification cycles for each sample. Some qPCR machines (for example the Bio-Rad CFX series) can be paused and opened to remove individual samples at different cycle numbers, which is helpful when amplifying different sample types at the same time.