Information

6.4A: Enrichment and Isolation - Biology


Learning Objectives

  • List the growth phases of microrganisms and the different types of growth media available to culture them

The most common growth media for microorganisms are nutrient broths and agar plates; specialized media are required for some microorganisms. Some, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.

Growth media: defined vs. undefined

An important distinction between growth media types is that of defined versus undefined media.

A defined medium will have known quantities of all ingredients. For microorganisms, this consists of providing trace elements and vitamins required by the microbe, and especially, a defined source of both carbon and nitrogen. Glucose or glycerol is often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources.

An undefined medium has some complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.

Types of media

Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally-rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45°C), which turns brown and gives the medium the color for which it is named.

Selective media are used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillinor tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. Media lacking an amino acid, such as proline in conjunction with E. coli unable to synthesize it, were commonly used by geneticists before the emergence of genomics to map bacterial chromosomes.

Differential/indicator media distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria. The agar triple-sugar iron (TSI) is one of the culture media used for the differentiation of most enterobacteria.

Growth in closed culture systems, such as a batch culture in LB broth, where no additional nutrients are added and waste products are not removed, the bacterial growth will follow a predicted growth curve and can be modeled.

Growth phases

During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.

Exponential phase (sometimes called the log or logarithmic phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population.Under controlled conditions, cyanobacteria can double their population four times a day. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with wastes.

The stationary phase is due to a growth-limiting factor; this is mostly depletion of a nutrient, and/or the formation of inhibitory products such as organic acids.

At death phase, bacteria run out of nutrients and die.

Culture

Batch culture is the most common laboratory-growth method in which bacterial growth is studied, but it is only one of many. The bacterial culture is incubated in a closed vessel with a single batch of medium.

In some experimental regimes, some of the bacterial culture is periodically removed and added to fresh sterile medium. In the extreme case, this leads to the continual renewal of the nutrients. This is a chemostat, also known as an open or continuous culture: a steady state defined by the rates of nutrient supply and bacterial growth. In comparison to batch culture, bacteria are maintained in exponential growth phase, and the growth rate of the bacteria is known. Related devices include turbidostats and auxostats. Bacterial growth can be suppressed with bacteriostats, without necessarily killing the bacteria.

In a synecological culture, a true-to-nature situation in which more than one bacterial species is present, the growth of microbes is more dynamic and continual.

Key Points

  • The most common growth media for microorganisms are nutrient broths and agar plates.
  • Open cultures allow for a replenishment of nutrients and a reduction of waste buildup in the media.
  • Selective media are used for the growth of only selected microorganisms.
  • Differential media or indicator media distinguish one microorganism type from another growing on the same media.

Key Terms

  • closed culture: A closed culture has no additional nutrients added to the system, and waste products are not removed. Cultures in a closed system will follow a predicted growth curve.
  • Enriched media: Contains nutrients required to support the growth of a wide variety of organisms.
  • open culture: A continuous culture where periodically some of the bacterial culture is removed and added to fresh sterile medium.

Recent Approaches in the Production of Novel Enzymes From Environmental Samples by Enrichment Culture and Metagenomic Approach

19.2 Production of Novel Enzymes From Environmental Samples by Enrichment Culture

Enrichment culture is basically an isolation technique designed to make conditions of growth very favorable for an organism of interest while having an unfavorable environment for any competition. It is the use of certain growth media to favor the growth of a particular microorganism over others, enriching a sample for the microorganism of interest. This enrichment is generally done by introducing nutrients or environmental conditions that only allow the growth of an organism of interest. Enrichment culture techniques are used to increase a small number of the desired organisms to detectable levels. This allows for the detection and identification of microorganisms with a variety of nutritional needs ( Liu et al., 2016 ). Enrichment culturing is often employed for isolating microbial species of interest from soil and marine habitats. The enrichment culturing includes two methods depending on type of media utilized: One is submerged (liquid medium), which is easy to control and optimize environmental and nutritional parameters, and the other is solid-state fermentation in which solid materials (raw or processed) are used as substrates ( Mahitha and Madhuri, 2016 ). Enrichment-dependent novel enzyme production in liquid fermentation method commonly involves the utilization of physical parameters like temperature, pH, incubation time, static and shaking conditions, and/or chemical properties like carbon and nitrogen sources and substrate and inoculum concentration as determining factors for the enzyme-producing isolates, thus providing the chance to grow and proliferate only the adapting species to the determining physical and/or chemical factor ( Shah and Patel, 2014 ). If the case is solid media utilization, certain factors have no influence as they are not maintained like static and stirring of the fermentation medium, and some new factors come into play as the substrate type (raw or processed) ( Figs. 19.1 and 19.2 ).

Fig. 19.1 . (A) Depolymerase activity indicated by formation of halo zone around the colony utilizing plastic as a nutrient source at the end of the seventh week. (B) Depolymerase activity in purified sample at the end of 1-week incubation.

Fig. 19.2 . Purification of asparaginase enzyme by gel-filtration chromatography.

The enrichment culture technique primarily employs substrates that are inexpensive (Jambul seeds agrowastes for enzymes like depolymerase, glutaminase, asparaginase, and bagasse distillery effluent—asparaginase dairy waste whey and fermented food waste for fibrinokinase) as the overall isolation process should be economical and repeatable until the target species is isolated ( Siddiqui et al., 2015 Vijayaraghavan et al., 2017 ). The enrichment technique also differs in the isolation procedure based on enzyme type like extracellular or intracellular in nature if extracellular, the presence of a soluble substrate is a universally utilized method as it gives the indication of enzyme activity through halo or clear-zone development that is easy to distinguish when compared with intracellular where it often requires destruction of the cell, addition of multiple reagents, and separate addition of substrate or secondary substrate to assess whether the target activity is present or not. The enrichment method of isolation was a powerful technique utilizing in many applications like selective enrichment, mimicking habitat to enrich microbes from extreme habitations in the search for many metabolites, antibiotics, and enzymes that are crucial in many sectors. The prospective effectiveness of the methods was multiplied manyfold by adding features like optimization of selecting method, innovations in fermentation technology, and secondary modifications of the initially selected organisms. The final active compound’s functionality can also be enhanced by increasing the purity of the compound by ammonium sulfate precipitation, dialysis, and chromatographic separation coupled with the determination of molecular weight using SDS-PAGE method. But, having said that, the fundamental disadvantage it encompasses is the dependency of the method on the cultivability of the microorganism being studied sadly, only a fraction (< 1%) of total microbial diversity in a given habitat is culturable, leaving out the rest being untapped ( Fig. 19.3 ).

Fig. 19.3 . Characterization of purified azoreductase enzyme by SDS-PAGE zymogram.


Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats.

We have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntington’s disease (HTT CAG repeat), Fragile X syndrome (FMR1 CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72 GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10 variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.


ProteoExtract ® Kits

The ProteoExtract ® kits cover the different steps in proteomics sample preparation from protein extraction and abundant protein removal to concentration of protein mixtures, removal of interfering substances, digestion of proteins, selective capturing of phosphorylated peptides, and selective enrichment for specific protein classes. All kits are cross-compatible.

  • Efficient and reproducible protein extraction
  • Protease inhibitor cocktails improve results in downstream analyses
  • Better spot resolution facilitated by nucleic acid digestion with protease-free Benzonase ® nuclease
  • Designed for compatibility with many applications including activity assays, Western blots, 1D and 2D PAGE, and mass spectrometry
  • Optimized protocols for different biological samples

Tips for Avoiding Isolation

During the ages of 20-30, you might find yourself experiencing a “quarter-life crisis.” Many young adults are still figuring out who they want to be and what they want to do with their lives. Intimate relationships can help to support you as you continue to explore your identity. If you find that you are isolated, take some time to assess why and how you can grow closer to the people in your life:

Talk to the people in your circle now about relationships and expectations. Open, honest conversations are the first step to a closer relationship.

If someone isn’t providing you with the things you need in a friendship, consider prioritizing your time on people who will better appreciate you.

Need to widen your circle? Join meetups or groups for people who share your interests. Don’t be afraid to reach out or set up a meetup of your own!

Reach out to a relationship therapist. Issues from earlier stages may leave you with mistrust, guilt, or feelings of inferiority. This can have a serious impact on your ability to be vulnerable with others and become part of a group. A relationship therapist can help you unravel these past experiences and move forward.


Isolation of Microbial Mutants: 4 Techniques | Microbiology

The following points highlight the top four techniques used for the isolation of microbial mutants. The techniques are: 1. Direct Observation 2. Enrichment Technique 3. Replica-Plating Technique 4. The Ames Test.

1. Direct Observation Technique:

In some cases, a colony growing on an agar plate can easily be seen to be different from the normal parental type (wild-type). For example, if the parental strain is pigmented, the observation of non-pigmented colonies may indicate the presence of mutants. Indicators can also be incorporated into the medium to detect microorganisms with and without particular metabolic capabilities.

For instance, pH indicators can be used into the medium to detect the production of acidic products. The indication of acid production by one microbial strain and not by other of the same microorganisms growing under identical conditions would show the presence of a mutant.

2. Enrichment Technique:

Enrichment technique is employed especially in isolating mutants resistant against phages, antibiotics, or toxic chemicals. Phage-resistant mutants can be isolated simply by plating the mutagenized, phenotypically expressed microbial population on plates containing phage particles.

Cells expressing the parental wild-type phenotype are killed only phage-resistant mutants develop into colonies. Such colonies are isolated. Similarly, mutants resistant to an antibiotic or a toxic chemical can be isolated by plating the microbial population with the antibiotic or the chemical.

3. Replica-Plating Technique:

Replica-plating technique is often used to isolate nutritional mutants (auxotrophs) as well as various other type of mutants, e.g., antibiotic resistant mutants.

For convenience, if one wants to isolate nutritional mutants employing replica-plating techniques (Fig. 29.13), he is required to follow the following steps:

(i) Bacterial cultures are diluted, and the cells are spread on the surface of semisolid nutrient agar medium in a Petri dish (called “master plate”). The medium in the master plate is a complete medium i.e., containing all the nutritional components required by the bacterial population. After a sufficient incubation period, each bacterium produces a visible colony on the surface of the agar in the master plate.

(ii) A piece of sterile velvet cloth is stretched over a cylindrical block of wood or metal that is slightly smaller in diameter than the Petri dishes used in the process.

(iii) The master plate is now inverted and gently pressed onto sterile velvet. Since the fibres of the velvet act as fine inoculating needle, some cells from each colony of the master plate stick to the velvet.

(iv) Other Petri dish (called “replica plate”) is taken containing a minimal medium i.e., a medium deficient with specific nutritional component,

(v) The replica-plate is now inverted and gently pressed onto the velvet thus stamping the bacterial cells onto the surface of its minimal medium. The replica plate is identically oriented at the application on the velvet with respect to mark placed on its rim so that the colonies that appear on the replica plate after incubation occupy positions congruent with those of their siblings on the master plate.

(vi) After sufficient incubation, it is observed that a colony that develops on the complete medium of the master plate fails to develop on the minimal medium of the master plate that lacks a specific nutritional component. Such colony is marked on the master plate and is isolated it represents mutant for that specific nutritional component not used in the minimal medium of the replica plate.

The replica-plating technique was developed by Joshua and Esther Lederberg in 1952 in order to provide direct evidence for the existence of pre-existing mutations originated spontaneously in microorganisms.

4. The Ames Test:

This test was developed by Ames and coworkers and is based on histidine-requiring (his – ) auxotrophic mutants of Salmonella typhimurium. Different his – mutants carry different types of mutations, i.e., transitions, transversions and frame- shifts.

In the Ames test, the frequency of reversion to his + (prototrophy) is scored in the especially constructed his – mutants. This is done by placing a known number of mutant cells on medium lacking histidine and scoring the number of colonies formed. The frequency of cells forming colonies gives the frequency of reversion. The frequency of spontaneous reversion to his + is quite rare, i.e., 10 -8 .

Ames test is routinely used to investigate the mutagenicity of various chemicals. Some of the chemicals may become mutagenic only when they are acted upon by liver enzymes.

For example, nitrates themselves are neither mutagenic nor carcinogenic. But in eukaryotic cells, nitrates are converted to introsamines, which are highly mutagenic and carcinogenic. In addition some chemicals may be mutagenic only to replicating DNA.

The routine Ames test addresses to both these needs as follows:

1. The his – cells are plated onto a medium that contains traces of histidine, which is enough to allow a few cell divisions, but inadequate for visible colony formation.

2. The test chemical is incubated with rat liver extract containing the liver enzymes, i.e., the microsomal fraction. This allows modification of the chemical in the same way as it would be in the liver of animals.

The procedure of Ames test is as follows. The his – bacterial cells are incubated with the liver extract, and then plated onto a medium containing traces of histidine this serves as the control plate.

The test plates contain the same medium but the his – cells are not treated with liver extract. The test chemical is treated with the rat liver extract and a filter paper disc is soaked in this solution. The filter paper disc is placed onto the medium of test plate (Fig. 29.14).

The chemical present in the filter paper acts on the his – cells growing in the test plate. The frequency of colonies formed in the control plate and the test plate are compared. An increase in the frequency in the case of test plate will indicate the test chemical to be mutagenic.

In order to increase the efficiency of the test, the his – strains used in the test are defective in DNA repair, and have increased permeability to chemicals. It has been observed that more than 90% of the chemicals that are mutagenic are also carcinogenic.


Protocol for Enrichment of mRNAs, excluding Globin mRNA, from Whole Blood Total RNA

This protocol describes the enrichment of poly(A) mRNA followed by globin mRNA & rRNA depletion (Section 1 and 2). The enriched RNA contains only mRNA (excluding globin) and not non-coding RNA. The enriched RNA is then used as input for directional RNA library preparation for sequencing on an Illumina instrument (Section 3).

This protocol requires the following NEBNext products:

  • NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490)
  • NEBNext Globin and rRNA Depletion Kit (Human/Mouse/Rat) with RNA Sample Purification Beads (NEB #E7755)
  • NEBNext Ultra II Directional RNA Library Prep for Illumina with Sample Purification Beads (NEB #E7765)

RNA Sample Requirements

RNA Integrity:
Assess the size and quality of the input RNA by running the RNA sample on an Agilent Bioanalyzer RNA 6000 Nano/Pico Chip to determine the RNA Integrity Number (RIN). For Poly(A) mRNA enrichment, high quality RNA with RIN Score >7 is required.

RNA Sample:
The RNA sample should be free of salts (e.g., Mg 2+ , or guanidinium salts) or organics (e.g., phenol and ethanol). RNA must be free of DNA. gDNA is a common contaminant in RNA preps. It may be carried over from the interphase of organic extractions or when the silica matrix of solid phase RNA purification methods is overloaded. If the total RNA sample may contain gDNA contamination, treat the sample with DNase I (not provided in this kit) to remove all traces of DNA. After treatment, the DNase I should be removed from the sample. Any residual DNase I may degrade the oligos necessary for the enrichment.

Input Amount:

This protocol has been tested with 100 ng human whole blood total RNA (DNA-free) in a maximum of 50 µl of nuclease-free water, quantified by an RNA-specific dye-assisted fluorometric method (Qubit ® ) and quality checked by Bioanalyzer.

Keep all buffers on ice, unless otherwise indicated.

1.0. Poly(A) mRNA Enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490)

1.1. Dilute the total RNA with nuclease-free water to a final volume of 50 &mul in a nuclease-free 0.2 ml PCR tube and keep on ice.

1.2. To wash the Oligo (dT) beads, add the components from the table below to a 1.5 ml nuclease-free tube. If preparing multiple libraries, beads for up to 10 samples can be added to a single 1.5 ml tube for subsequent washes (use magnet NEB #S1506 for 1.5 ml tubes). The purpose of this step is to bring the beads from the storage buffer into the binding buffer. The 2X Binding Buffer does not have to be diluted for this step.

NEBNext RNA Binding Buffer (2X)

Total volume

1.3. Wash the beads by pipetting up and down six times.

1.4. Place the tube on the magnet and incubate at room temperature until the solution is clear (

1.5. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

1.6. Remove the tube from the magnetic rack.

1.7. Add 100 &mul RNA Binding Buffer (2X) to the beads and wash by pipetting up and down six times. If preparing multiple libraries, add 100 µl RNA Binding Buffer (2X) per sample. The Binding Buffer does not have to be diluted.

1.8. Place the tubes on the magnet and incubate at room temperature until the solution is clear (

1.9. Remove and discard the supernatant from the tube. Take care not to disturb the beads.

1.10. Add 50 &mul RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous. If preparing multiple libraries, add 50 &mul RNA Binding Buffer (2X) per sample.

1.11. Add 50 &mul beads to each RNA sample from Step 1.1 Mix thoroughly by pipetting up and down six times. This binding step removes most of the non-target RNA.

1.12. Place the tube in a thermocycler and close the lid. Heat the sample at 65°C for 5 minutes and cool to 4°C with the heated lid set at &ge 75°C. This step will denature the RNA and facilitate binding of the mRNA to the beads.

1.13. Remove the tube from the thermocycler when the temperature reaches 4°C.

1.14. Mix thoroughly by pipetting up and down six times. Place the tube on the bench and incubate at room temperature for 5 minutes to allow the mRNA to bind to the beads.

1.15. Place the tube on the magnetic rack at room temperature until the solution is clear (

1.16. Remove and discard all of the supernatant. Take care not to disturb the beads.

1.17. Remove the tube from the magnetic rack.

1.18. To remove unbound RNA add 200 &mul of Wash Buffer to the tube. Gently pipette the entire volume up and down 6 times to mix thoroughly.

1.19 Spin down the tube briefly to collect the liquid from the wall and lid of the tube.

Note: It is important to spin down the tube to prevent carryover of the Wash Buffer in subsequent steps.

1.20. Place the tube on the magnetic rack at room temperature until the solution is clear (

1.21. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads containing the mRNA.

1.22. Remove the tube from the magnetic rack.

1.24. Add 11 &mul of nuclease-free water to each tube. Gently pipette up and down 6 times to mix thoroughly.

1.25. Place the tube in the thermocycler. Close the lid and heat the samples at 80°C for 2 minutes, then cool to 25°C with the heated lid set at &ge 90°C to elute the mRNA from the beads.

1.26. Remove the tube from the thermocycler when the temperature reaches 25°C.

1.27 Immediately place the tube on the magnet at room temperature until the solution is clear (

1.28. Collect the purified mRNA by transferring 10 &mul of the supernatant to a clean nuclease-free PCR tube.

1.29. Place the RNA on ice and proceed to the Globin and rRNA Depletion in Section 2.

2.0. Globin and rRNA Depletion using the NEBNext Globin and rRNA Depletion Kit (NEB #E7750/E7755)

2.1 Probe Hybridization to RNA

2.1.2. Assemble the following RNA/Probe hybridization reaction on ice:

RNA/PROBE HYBRIDIZATION REACTION

(white) mRNA in nuclease-free water (Step 1.29)

(white) NEBNext Globin and rRNA Depletion Solution

(white) NEBNext Probe Hybridization Buffer

Total Volume

2.1.3. Mix thoroughly by gently pipetting up and down at least 10 times. Note: It&rsquos crucial to mix well at this step.

2.1.4. Briefly spin down the tube in a microcentrifuge to collect the liquid from the side of the tube.

2.1.5. Place the tube in a pre-heated thermocycler and run the following program with the heated lid set at 105°C. This program will take approximately 15&ndash20 minutes to complete:

2.1.6. Briefly spin down the tube in a microcentrifuge, and place on ice. Proceed immediately to the RNase H digestion.

2.2. RNase H Digestion

2.2.1. Assemble the following RNase H digestion reaction on ice:

(white) NEBNext Thermostable RNase H

(white) RNase H Reaction Buffer

Total Volume

2.2.2. Mix thoroughly by gently pipetting up and down at least 10 times.

2.2.3. Briefly spin down the tube in a microcentrifuge.

2.2.4. Incubate the tube in a pre-heated thermocycler for 30 minutes at 50°C with the lid set at 55°C.

2.2.5. Briefly spin down the tube in a microcentrifuge, and place on ice. Proceed immediately to the DNase I digestion.

2.3. DNase I Digestion

2.3.1. Assemble the following DNase I digestion reaction on ice:

DNASE I DIGESTION REACTION

RNase H treated RNA (Step 1.2.5)

(white) DNase I Reaction Buffer

(white) NEBNext DNase I

Total Volume

2.3.2. Mix thoroughly by pipetting up and down at least 10 times.

2.3.3. Briefly spin down the tube in a microcentrifuge.

2.3.4. Incubate the tube in a pre-heated thermocycler for 30 minutes at 37°C with the lid set at 40°C or off.

2.3.5. Briefly spin down the tube in a microcentrifuge, and place on ice. Proceed immediately to the RNA Purification step.

2.4. RNA Purification Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads

2.4.1. Vortex the Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads to resuspend.

2.4.2. Add 90 &mul (1.8X) beads to the RNA Sample from Step 2.3.5 and mix thoroughly by pipetting up and down at least 10 times.

2.4.3. Incubate the tube for 15 minutes on ice to bind the RNA to the beads.

2.4.4. Place the tube on a magnetic rack to separate the beads from the supernatant.

2.4.5. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads which contain the RNA.

2.4.6. Add 200 µl of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads, which contain the RNA.

2.4.7. Repeat Step 2.4.6 once for a total of two washes.

2.4.8. Completely remove residual ethanol, and air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of RNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

2.4.9. Remove the tube from the magnetic rack. Elute the RNA from the beads by adding 7 &mul of nuclease-free water. Mix thoroughly by pipetting up and down at least 10 times and briefly spin the tube.

2.4.10. Incubate the tube for 2 minutes at room temperature.

2.4.11. Place the tube on the magnetic rack until the solution is clear (

2.4.12. Remove 5 µl of the supernatant containing RNA and transfer to a nuclease-free tube.

2.4.13. Place the tube on ice and proceed with RNA-Seq library construction (protocol below) or other downstream application. Alternatively, the sample can be stored at -80°C.

Note: The next step provides a fragmentation incubation time resulting in an RNA insert of

200nt. Refer to Appendix (Section 4 of the NEBNext Ultra II Directional RNA Library Prep for Illumina Manual) for fragmentation conditions if you are preparing libraries with large inserts (>200 bp).

3.1.3. Incubate the sample for 15 minutes at 94°C in a thermocycler with the heated lid set at 105°C.

3.1.4. Immediately transfer the tube to ice for 1 minute.

3.1.5 Perform a quick spin to collect all liquid from the sides of the tube and proceed to First Strand cDNA Synthesis.

3.2. First Strand cDNA Synthesis

3.2.1. Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented
and primed RNA from Step 3.1.5:

FIRST STRAND SYNTHESIS REACTION

Fragmented and primed RNA (Step 3.1.5)

(brown) NEBNext Strand Specificity Reagent

(lilac) NEBNext First Strand Synthesis Enzyme Mix

Total Volume

3.2.2. Mix thoroughly by pipetting up and down 10 times.

3.2.3. Incubate the sample in a preheated thermocycler with the heated lid set at &ge 80°C as follows:

Note: If you are following recommendations in Section 4 of the NEBNext Ultra II Directional RNA Library Prep for Illumina Manual for libraries with longer inserts (>200 bases), increase the incubation at 42°C from 15 minutes to 50 minutes at Step 2 below.

3.2.4. Proceed directly to Second Strand cDNA Synthesis.

3.3. Second Strand cDNA Synthesis

3.3.1. Assemble the second strand cDNA synthesis reaction on ice by adding the following components into the first strand synthesis product from Step 3.2.4.

SECOND STRAND SYNTHESIS REACTION

First-Strand Synthesis Product (Step 3.2.4)

(orange) NEBNext Second Strand Synthesis Reaction Buffer
with dUTP Mix (10X)

(orange) NEBNext Second Strand Synthesis Enzyme Mix

Total Volume

3.3.2. Keeping the tube on ice, mix thoroughly by pipetting up and down at least 10 times.

3.3.3. Incubate in a thermocycler for 1 hour at 16°C with the heated lid set at &le 40°C (or off).

3.4. Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads

3.4.1. Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.

3.4.2. Add 144 &mul (1.8X) of resuspended beads to the second strand synthesis reaction (

80 &mul). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

3.4.3. Incubate for 5 minutes at room temperature.

3.4.4. Briefly spin the tube in a microcentrifuge to collect any sample on the sides of the tube. Place the tube on a magnetic rack to separate beads from the supernatant. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads, which contain DNA. Caution: do not discard beads.

3.4.5. Add 200 &mul of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

3.4.6. Repeat Step 3.4.5 once for a total of 2 washing steps.

3.4.7. Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

3.4.8. Remove the tube from the magnetic rack. Elute the DNA from the beads by adding 53 &mul 0.1X TE Buffer (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down at least 10 times. Quickly spin the tube and incubate for 2 minutes at room temperature. Place the tube on the magnetic rack until the solution is clear.

3.4.9. Remove 50 µl of the supernatant and transfer to a clean nuclease-free PCR tube.

Note: If you need to stop at this point in the protocol samples can be stored at &ndash20°C.

3.5. End Prep of cDNA Library

3.5.1. Assemble the End Prep reaction on ice by adding the following components to the second strand synthesis product from
Step 3.4.9.

Second Strand cDNA Synthesis Product (Step 3.4.9)

(green) NEBNext Ultra II End Prep Reaction Buffer

(green) NEBNext Ultra II End Prep Enzyme Mix

Total Volume

If a master mix is made, add 10 µl of master mix to 50 µl of cDNA for the End Prep reaction.

3.5.2. Set a 100 &mul or 200 &mul pipette to 50 &mul and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.

3.5.3. Incubate the sample in a thermocycler with the heated lid set at &ge 75°C as follows.

3.5.4. Proceed immediately to Adaptor Ligation.

3.6. Adaptor Ligation

Note: If you are selecting for libraries with larger insert size (>200 nt) follow the size selection recommendations in Appendix, Section 4 of the NEBNext Ultra II Directional RNA Library Prep for Illumina Manual.

3.7.1. Add 87 &mul (0.9X) resuspended SPRIselect Beads or NEBNext Sample Purification Beads and mix well on a vortex mixer or by pipetting up and down at least 10 times.

3.7.2. Incubate for 10 minutes at room temperature.

3.7.3. Quickly spin the tube in a microcentrifuge and place the tube on a magnetic rack to separate beads from the supernatant. After the solution is clear (

5 minutes), discard the supernatant that contains unwanted fragments. Caution: do not discard beads.

3.7.4. Add 200 &mul of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

3.7.5. Repeat Step 3.7.4 once for a total of 2 washing steps.

3.7.6. Briefly spin the tube and put the tube back in the magnetic rack.

3.7.7. Completely remove the residual ethanol, and air-dry beads until the beads are dry for up to 5 minutes while the tube is on the magnetic rack with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

3.7.8. Remove the tube from the magnetic rack. Elute DNA target from the beads by adding 17 &mul 0.1X TE (provided) to the beads. Mix well on a vortex or by pipetting up and down. Quickly spin the tube and incubate for 2 minutes at room temperature. Place the tube in the magnet until the solution is clear.

3.7.9. Without disturbing the bead pellet, transfer 15 &mul of the supernatant to a clean PCR tube and proceed to PCR enrichment.

Note: If you need to stop at this point in the protocol, samples can be stored at &ndash20°C.

3.8. PCR Enrichment of Adaptor Ligated DNA

Check and verify that the concentration of your oligos is 10 &muM on the label.

Use Option A for any NEBNext Oligos kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes.

Use Option B for any NEBNext Oligos kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse primers (i7 and i5) combined.

3.8.1. Set up the PCR reaction as described below based on the type of oligos (PCR primers) used.

3.8.1A. Forward and Reverse Primers Separate

Adaptor Ligated DNA (Step 2.11.9)

(blue) NEBNext Ultra II Q5 ® Master Mix

Universal PCR Primer/i5 Primer*,**

Total Volume

* NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual
for determining valid barcode combinations.

** Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample.

3.8.1B. Forward and Reverse Primers Combined

Adaptor Ligated DNA (Step 2.11.9)

(blue) NEBNext Ultra II Q5 Master Mix

Index (X) Primer/i7 Primer Mix*

Total Volume

* NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual
for determining valid barcode combinations.

** Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample

3.8.2. Mix well by gently pipetting up and down 10 times. Quickly spin the tube in a microcentrifuge.

3.8.3. Place the tube on a thermocycler with the heated lid set to 105°C and perform PCR amplification using the following PCR cycling conditions (refer to Table 3.8.3A and Table 3.8.3B):

* The number of PCR cycles should be adjusted based on RNA input.

** It is important to limit the number of PCR cycles to avoid overamplification.
If overamplification occurs, a second peak

1,000 bp will appear on the Bioanalyzer trace (See Figure 5.2 of the NEBNext Ultra II Directional RNA Library Prep for Illumina Manual).

Table 3.8.3B: Recommended PCR cycles based on total RNA input amount:

* The PCR cycles are recommended based on high quality human whole blood total RNA. To prevent over-amplification, the number of cycles may require optimization based on the sample quality and the fraction of globin mRNA. For RNA where globin mRNA is > than 50% of the transcripts (once rRNA is removed), follow the higher cycle recommendation for that input.

3.9. Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads

3.9.1. Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.

3.9.2. Add 45 &mul (0.9X) of resuspended beads to the PCR reaction (

50 &mul). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

3.9.3. Incubate for 5 minutes at room temperature.

3.9.4. Quickly spin the tube in a microcentrifuge and place the tube on a magnetic rack to separate beads from the supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. Caution: do not discard beads.

3.9.5. Add 200 &mul of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

3.9.6. Repeat Step 3.9.5 once for a total of 2 washing steps.

3.9.7. Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

3.9.8. Remove the tube from the magnetic rack. Elute the DNA target from the beads by adding 23 &mul 0.1X TE (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down ten times. Quickly spin the tube in a microcentrifuge and incubate for 2 minutes at room temperature. Place the tube in the magnetic rack until the solution is clear.

3.9.9. Transfer 20 &mul of the supernatant to a clean PCR tube and store at &ndash20°C.

3.10. Library Quantification

3.10.1. Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries.

3.10.2. Check that the electropherogram shows a narrow distribution with a peak size approximately 300 bp.

80 bp (primers) or 128 bp (adaptor-dimer) is visible in the bioanalyzer traces, bring up the sample volume (from Step 3.9.9) to 50 &mul with 0.1X TE buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead Cleanup Step (Section 3.9).

Figure 3.9.1 Example of RNA library size distribution on a Bioanalyzer.


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Trifanny Yeo and Swee Jin Tan: These authors contributed equally to this work.

Affiliations

Clearbridge Accelerator Pte Ltd, 81 Science Park Drive, The Chadwick, #02-03, Singapore Science Park 1, Singapore, 118257, Singapore

Trifanny Yeo & Swee Jin Tan

School of Biological Science, National Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore

Cancer Therapeutics Research Laboratory, National Cancer Centre Singapore, 11 Hospital Drive, Singapore, 169610, Singapore

Dawn Ping Xi Lau, Sai Sakktee Krisna, Gopal Iyer & Daniel S.W. Tan

Department of Pathology, Singapore General Hospital, Outram Road, Singapore, 169608, Singapore

Yong Wei Chua, Gek San Tan & Tony Kiat Hon Lim

Cancer Stem Cell Biology, Genome Institute of Singapore, 60 Biopolis St, #02-01, 138672, Singapore

Division of Medical Oncology, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610, Singapore

Daniel S.W. Tan & Wan-Teck Lim

Duke-NUS Medical School, 8 College Road, 169857, Singapore

Institute of Molecular and Cell Biology, A*Star, 61 Biopolis Drive Proteos, 138673, Singapore

Department of Biomedical Engineering, National University of Singapore, 4 Engineering Drive 3, Block E4, #04-08, Singapore 117583, Singapore

Mechanobiology Institute of Singapore, 5A Engineering Drive 1, Singapore 117411, Singapore


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