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StatusPublic on Aug 25,
TitleIRES_Screening_Rep1_Bin7
Sample typeSRA
 
Source nameDesigned oligonucleotides
Organismsynthetic construct
Characteristicsfunctional assay: circRNA cap-independent translation
cell line: HEKT
master plasmid: oligo-split-eGFP-circRNA
Extracted moleculegenomic DNA
Extraction protocolIRES screening: Total DNA from each expression bin was extracted using Quick-DNA Microprep Plus Kit (Zymo Research).Synthesis (NEB BioLabs), and purified with DNase digestion (TURBO DNase; Thermo Fisher Scientific) and RNase R digestion (MACLAB; 20 U RNase R per 20 μg of RNA).
Polysome profiling: The total RNAs of each fraction were purified respectively using RNeasy Mini Kit (Qiagen) with DNase treatment (Qiagen). The RNAs were then treated with RNase R and purified using RNA Clean & Concentrator (Zymo Research).
M2-seq: The circRNA of oligo of interest was produced with in vitro transcription using HiScribe™ T7 High Yield RNA
IRES screening: Three rounds of PCR using NEBNext Ultra II Q5 Master Mix (NEB BioLabs) were performed to generate the library for next-generation sequencing. For the first PCR, oligo library sequence was amplified using the following primer set – Fw: GGGATCACTCTCGGCATGGA; Rv: GCTCCTCGCCCTTGCTCAC. Each 50 μL PCR reaction contained ng total DNA, nM forward primer, and nM reverse primer. The parameters for the PCR were 98°C for 1 min, 24 cycles of 98°C for 30 s, 65°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. For the second PCR, adapter priming sequences were added to the oligo library using the following primer set – Fw: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGATCACTCTCGGCATGGA; Rv: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTCCTCGCCCTTGCTCAC (underlined sequence represents the sequence for illumina adapters priming). Each 50 μL PCR reaction contained μL of the first PCR product, nM forward primer, and nM reverse primer. The parameters for the PCR were 98°C for 1 min, 18 cycles of 98°C for 30 s, 70°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. For the third PCR, custom barcodes adapters were used for the reaction. Each 50 μL PCR reaction contained 1 μL of diluted second PCR product, 1 μM Ad1 adapter, and 1 μM Ad2 adapter. The parameters for the PCR were 98°C for 1 min, 15 cycles of 98°C for 30 s, 72°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. The PCR product was then separated on a 2% E-Gel™ EX Agarose Gel (Invitrogen) and the DNA fragments with expected size were purified using Zymoclean Gel DNA Recovery Kit (Zymo Research).
Polysome profiling: The oligo sequences from each sample were amplified by RT-PCR with SuperScript™ IV One-Step RT-PCR System (Thermo Fisher Scientific) using primers flanking the oligo sequence (Fw: GGGATCACTCTCGGCATGGA; Rv: GCTCCTCGCCCTTGCTCAC). cDNA libraries were generated and sequenced with HiSeq using the library generation method as for IRES screening.
M2-seq: The complimentary single-stranded DNA of the circRNA was generated by reverse transcription using SuperScript™ IV (Thermo Fisher Scientific) with oligo sequence specific primer (TCTTTTGTTGGAGTCTACTCGACTCCTTT) and purified with DNA Clean & Concentrator-5 (Zymo Research). Three rounds of PCR using NEBNext Ultra II Q5 Master Mix (NEB BioLabs) were performed to generate the cDNA library for next-generation sequencing. For the first PCR, oligo library sequence was amplified using the following primer set – Fw: GAACGACTCGAGTAGAGTCGAAAA; Rv: TCTTTTGTTGGAGTCTACTCGACTCCTTT. Each 50 μL PCR reaction contained ng total DNA, nM forward primer, and nM reverse primer. The parameters for the PCR were 98°C for 1 min, 15 cycles of 98°C for 30 s, 67°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. For the second PCR, sequences for adapters priming were added to the oligo library using the following primer set – Fw: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAACGACTCGAGTAGAGTCGAAAA; Rv: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTTTTGTTGGAGTCTACTCGACTCCTTT (underline represents the sequence for illumina adapters priming). Each 50 μL PCR reaction contained μL of the first PCR product, nM forward primer, and nM reverse primer. The parameters for the PCR were 98°C for 1 min, 12 cycles of 98°C for 30 s, 67°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. For the third PCR, custom barcodes adapters were used for the reaction. Each 50 μL PCR reaction contained 1 μL of diluted second PCR product, 1 μM Ad1 adapter, and 1 μM Ad2 adapter. The parameters for the PCR were 98°C for 1 min, 15 cycles of 98°C for 30 s, 72°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. The PCR product was then separated on a 2% E-Gel™ EX Agarose Gel (Invitrogen) and the DNA fragments with expected size were purified using Zymoclean Gel DNA Recovery Kit (Zymo Research).
IRES screening: DNA-seq.
Polysome profiling: RNA-seq
M2-seq: RNA-seq
 
Library strategyOTHER
Library sourcegenomic
Library selectionother
Instrument modelIllumina MiSeq
 
DescriptionTotal DNA
Oligo_eGFP_expression.xlsx
Data processingIRES screening and polysome profiling: Single-end reads in the length of nt were trimmed to nt containing the unique oligo variable region using fastx_trimmer module (-f 54). Trimmed reads were mapped to the synthetic library sequences using Bowtie2 with default settings. Multi-mappers were excluded from the subsequent analysis by excluding the reads with the "XS" flag in the .sam files (grep -v). The number of reads for each designed oligo was counted in each bin respectively using bedtools multiBamCov module.
M2-seq: Single-end reads in the length of nt were trimmed to nt containing the unique oligo sequence using fastx_trimmer module (-f 39 -l ). ShapeMapper was used to align the trimmed reads to reference sequences and record mutations, and the results were converted to M2-seq data and mutation spectra using scripts available at https://github.com/ribokit/m2seq. The M2-net analysis, mutate-and-map visualization, RNA structural analysis, and RNA structure visualization were performed as described in Cheng et al. (PNAS, ).
Genome_build: Synthetic library oligonucleotides
Supplementary_files_format_and_content: IRES screening: .xlsx file that contains the synthetic oligo index, sequence, and cap-independnet translation activity.
Supplementary_files_format_and_content: Polysome profiling: .xlsx file that contains the number of (poly)ribosome-enriched oligos
Supplementary_files_format_and_content: M2-seq: .rdat file for 2nd structure determination
 
Submission dateJun 23,
Last update dateAug 25,
Contact nameChun-Kan Chen
E-mail(s)[email protected]
Organization nameStanford University
Street addressCCSR , Campus Drive
CityStanford
State/provinceCA
ZIP/Postal code
CountryUSA
 
Platform IDGPL
Series (1)
GSEStructured elements drive extensive circular RNA translation
Relations
BioSampleSAMN
SRASRX

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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Value Oligos

Upload up to oligo sequences at one time, using the Excel spreadsheet template, below.   Enter names and sequences into the template, save as an XLS, XLSX, CSV, or TXT file, and upload when complete.  

   Click below to download the Excel template to your local computer

* Sequences should be between 5 and 40 bases and must consist of A, G, C and T only.



   Upload your saved sequence file here

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   Select the options below to apply to your imported sequences.

Synthesis Scale *Purification*Format *Delivery *  

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Fisher thermo custom oligos

4 tips for accurate oligonucleotide quantification using Thermo Scientific™ NanoDrop™ instruments

Due to their ease of use, fast speed, and accuracy, NanoDrop instruments (Thermo Fisher Scientific) are frequently used to perform quantification on various types of nucleic acid molecules, including DNA oligonucleotides, double-stranded DNA, and RNA. Specifically when measuring oligonucleotides, it is important to acknowledge their unique characteristics, which can require adjustment of NanoDrop parameters for correct readings.

Experimental collaboration by IDT and Thermo Fisher Scientific scientists has resulted in a Technical report: Accurate oligonucleotide quantification using Thermo Scientific NanoDrop instruments. Here, we provide a summary of recommended procedural adjustments, when using the NanoDrop instrument to obtain accurate quantification of oligonucleotides.

1. Use an oligo-specific conversion factor instead of the general single-stranded DNA (ssDNA) conversion factor of 33 μg/A.

Oligonucleotides are short, single-stranded molecules. Their UV spectrum and extinction coefficient are much more dependent on their base composition and sequence context compared to longer ssDNA and RNA molecules. Thus, for accurate quantification, determine and apply an oligo-specific conversion factor. The NanoDrop software will do this for you, when you choose either “custom” or “oligo” option from the sample type menu.

2. Use the MicroArray module of the NanoDrop software for measuring oligonucleotides with modifications.

Many of the modifications added to the 5′ or 3′ ends of oligonucleotides absorb light, and as a result, can affect quantification results. Thus, for accurate quantification of modified oligonucleotides, use a correction factor. To obtain this value with the NanoDrop software, use the MicroArray module, which provides an automatic correction for modification absorbance.

3. Make dilutions to ensure absorbance measurements use 1 or mm path lengths.

NanoDrop instruments can measure a wide concentration range of nucleic acids through use of multiple path lengths. However, an instrument’s acceptable error increases as the path length is shortened. The most accurate quantification results can be achieved by measuring diluted oligonucleotides with an absorbance < This will ensure the instrument makes absorbance measurements at a 1 or mm path length, providing a much smaller error tolerance.

Determine the approximate absorbance of an oligonucleotide stock solution using the Beer-Lambert equation:

A = ε b c

Where:
A = Absorbance
ε = Molar attenuation coefficient (L/(mole·cm), obtained from the manufacturer)
b = Path length (cm)
c = Concentration (M, mole/L)

4. Turn off the default baseline correction for oligonucleotides with modifications that absorb light at nm.

The default setting for NanoDrop instrument measurements is nm. This correction is necessary, because it adjusts for any light scattering event that may skew results. However, certain oligonucleotide modifications absorb light at this wavelength, so this correction could lead to an inaccurate result.

It is also noteworthy to mention that traditional purity ratios (A/ and A/), used as an indication of the presence of various contaminants in nucleic acid samples, do not apply to oligonucleotides, because the shape of the oligonucleotide UV spectrum is highly dependent on base composition.

Obtain more detailed explanations for why and how to make these adjustments in the Technical report: Accurate oligonucleotide quantification using Thermo Scientific NanoDrop instruments.

Author(s)

Bahri Karaçay, PhD, Commercial Marketing Manager, CRISPR/Functional Genomics, IDT.

Sours: https://www.idtdna.com/pages/education/decoded/article/4-tips-for-accurate-oligonucleotide-quantification-using-thermo-scientific-nanodrop-instruments
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