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Novex tbe urea gel

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The Novex TBE-Urea gel is a pre-cast polyacrylamide gel designed for high-resolution separation of nucleic acid samples under denaturing conditions. The gel is formulated with a Tris-Borate-EDTA (TBE) buffer and contains urea to maintain the denaturing environment, enabling the analysis of single-stranded DNA and RNA samples.

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42 protocols using novex tbe urea gel

1

Small RNA-Seq Library Preparation and Analysis

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Small RNA-seq libraries were constructed as described previously (Lu et al., 2007 (link)), with minor modifications. Total RNA was isolated using the mirVana miRNA Isolation Kit (Ambion). Small RNAs (20–30 nt) were separated on a 15% Novex TBE-Urea gel (Invitrogen) and purified. The purified small RNAs were ligated first with the 5′ RNA adapter and then with the 3′ RNA adapter provided in the TruSeq Small RNA Library Prep Kit (Illumina). At each step, the ligated product was subjected to polyacrylamide gel electrophoresis (PAGE) and gel purified. After first-strand synthesis and 11 cycles of PCR amplification, the product was separated by PAGE, gel purified, and submitted for sequencing on the Illumina NextSeq500 platform. Adapter sequences were removed from the raw sequences using Trimommatic (v.0.33), and then the cleaned sequences were mapped onto the Arabidopsis reference genome (TAIR10) using bowtie2 (v.2.2.2). The quantification and statistical analysis of mapped reads were carried out using HTSeq (Kim et al., 2013 (link); Anders et al., 2015 (link)) and edgeR (Robinson et al., 2010 (link)) packages, respectively.
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2

RNA Extraction and Small RNA Sequencing

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Total RNA was isolated from the fresh-frozen tissues and 10-μm thick paraffin-embedded tissue sections using the RecoverAll™ Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. The quality and quantity of the extracted total RNA were analyzed with an ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). In addition, the 2100 Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany) was used for the estimation of the RNA integrity number (RIN) score.
Small RNAs (20–30 nt) were purified from 15% Novex TBE-Urea Gel (Invitrogen) and ligated first with the 5’ RNA adaptor and then with the 3’ RNA adaptor provided by Illumina TruSeq small RNA sample preparation protocol. In each step, the ligated product was PAGE-gel purified. After first-strand synthesis and 11 cycles of PCR amplification, the product was PAGE-gel purified and submitted for sequencing on an Illumina NextSeq500 at LAS (Seoul, Korea; https://www.las.kr/).
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3

Quantifying Steady-State mt-tRNA Levels

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To study steady-state levels of mt-tRNAs, 1.2 μg of total cell RNA from BMDMs was resolved in a 10% Novex TBE-Urea gel in 1× TBE buffer (Invitrogen). The RNA was then transferred onto a Hybond-N+ Nylon membrane (GE Healthcare) at 30 V for 1 hour in 0.5× TBE. RNA was cross-linked to the membrane with 1.5 J/cm2 of ultraviolet radiation. Oligonucleotides for detecting mt-tRNAALA (5′-GACTTCATCCTACATCTATTG-3′) and mt-tRNACYS (5′-TCTCTACACCTTCGAATTTG-3′) were radiolabeled with [γ-32P]-ATP at the 5′ end using T4 polynucleotide kinase (New England Biolabs). To detect the 5.8S cytosolic rRNA (loading control), 5.8S rRNA was PCR amplified and gel purified (Qiagen). The dsDNA obtained was used as a template to prepare the DNA probe with radioactive [α-32P]-dCTP using the Prime-It II random primer labeling kit (Agilent). Probe hybridization temperatures for mt-tRNAs and 5.8S rRNA were 42°C and 65°C, respectively. Hybridized membranes were exposed on a phosphorimager screen and band intensity was quantified using ImageJ (NIH).
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4

Northern Blot for RNA Analysis

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Northern blot was performed similarly as before72 (link),73 (link). Briefly, 1–5 μg purified total RNA or 5 pmole synthetic RNA oligos were resolved on 15% Novex TBE-Urea gel (Invitrogen #EC6885BOX) and transferred to Amersham Hybond-N + nylon membrane (Cytiva #RPN203B) by Trans-Blot SD semi-dry transfer apparatus (Bio-Rad). The membrane was cross-linked with 254 nm wavelength by Stratalinker (Strategene).
For Northern blot: After UV crosslinking, the membrane was blocked by ExpressHyb Hybridization Solution (Takara Bio #636833) and probed with biotinylated DNA probe (probe sequences see Supplementary Table 7) following the manufacturer’s instructions. Notably, the upper (U6 and full-length tRNA) and lower (tRF) parts of the membrane was cut after transfer, probed, and developed separately to avoid saturation of tRNA signals. Hybridized membrane was detected with Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher #89880).
For immuno-Northern blot: After UV crosslinking, the membrane was blocked with 3% milk in PBST and detected by m1A primary antibody (MBL #D3453, used at 1:2000 dilution) followed by anti-mouse HRP-linked secondary antibody (Cell Signaling #7076, used at 1:5000 dilution). Chemiluminescence detection was performed with Immobilon HRP substrate (Millipore #WBKLS0500). Oligo size was compared to microRNA marker (NEB #N2102S) on the gel.
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5

Ring-nuclease-mediated cOA4 processing

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Increasing concentrations of both ring-nucleases from 10 nM to 150 nM were incubated with 25 nM of cOA4 at 70 °C for 30 min followed by addition of 18 nM of SisCsx1 and 2.5 μΜ of RNA1 and further incubation at 70 °C for 5 min. The reactions were then separated using 15% Novex TBE-urea gel (Invitrogen).
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6

Bacterial RNA Extraction and Purification

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E. coli BW25113 strain was grown at 37 °C until OD600 ~0.4–0.6. For extraction of total RNA, cells were resuspended in ice-cold buffer (0.3 M NaOAc pH 4.5, 10 mM EDTA) followed by direct phenol extraction using cold acid phenol/chloroform (5:1, pH 4.5). RNA was extracted from the resulting pellet by dissolving the pellet in ice-cold buffer (10 mM NaOAc pH 4.5, 800 mM LiCl). Ribosomal RNA was further depleted from the sample via three consecutive precipitations with 0.2 volumes of 2-propanol, followed by a fourth precipitation with 1 volume of 2-propanol. RNA that was obtained in the third (fraction A) and fourth (fractions B–D) precipitation steps was size selected on a 10% Novex TBE–urea gel (Invitrogen) as indicated in Figure S1b.
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7

Northern Blot Analysis of Mvh Expression

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Total RNA was extracted from adult testes (wild-type and Mvh homozygote) and organ cultured tissues (4-day culture) using Sepasol-RNAI SuperG (Nacalai Tesque) according to the manufacturer’s manual. A total of 20 μg of total RNA was electrophoresed on a 10% NovexTM TBE-Urea Gel (Invitrogen) and transferred to a Hybond-N+ membrane (Amersham Biosciences) using semi-dry electroblotter at 400mA for 30 min and subsequently UV crosslinked. Oligo DNA probes (10 ng each) were labeled using gamma [32P]-ATP with a MEGALABEL Kit (Takara-Shuzo). Hybridization was performed in a hybridization buffer (10% SDS, 10% dextran sulfate, 1M NaCl, 0.5mg/ml sonicated salmon sperm DNA) at 65℃ overnight. The membranes were then washed twice in 2xSSC and 0.5% SDS at room temperature for 30min and in 0.2xSSC and 0.5% SDS at 65℃ for 30 min. Next, the signals were visualized using a BAS-3000 image-analyzer (GE-HealthCare).
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8

Northern Blot Analysis of RNA

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A total of 20 μg of total RNA was electrophoresed on a 10% NovexTM TBE-Urea Gel (Invitrogen) and transferred to a Hybond-N+ membrane using a semi-dry electroblotter at 400 mA for 30 min, followed by UV crosslinking. Oligo DNA probes (10 ng each) were labeled with gamma [32P]-ATP using a MEGALABEL Kit (Invitrogen). Hybridization was performed in hybridization buffer (10% SDS, 10% dextran sulfate, 1 M NaCl, 0.5 mg/mL sonicated salmon sperm DNA) at 65 °C overnight. The membranes were washed twice in 2x SSC and 0.5% SDS at room temperature for 30 min and in 0.2x SSC and 0.5% SDS at 65 °C for 30 min. Next, the signals were visualized with a BAS-3000 image-analyzer (GE Healthcare, Little Chalfont, UK).
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9

Cas12a Family-Mediated DNA Cleavage

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In-vitro digestion reactions were carried out with three different types of the Cas12a family (LbCas12a, AsCas12a, and FnCas12a were purchased from New England Biolabs Inc. or purified in the lab, Integrated DNA Technologies Inc., and abm®, respectively) and a wide array of modified crRNAs (purchased from DNA Technologies Inc.). Cas12a and crRNA were mixed with a 1:1 ratio (100 nM:100 nM) in 1× NEBuffer 2.1 and pre-incubated at 25 °C for 15 min to promote the ribonucleoprotein complex formation. DNA activator (final concentration of 7 nM) was then added to the mixture to produce a total volume of 30 μl and incubated at 37 °C for 30 min19 (link). The sample was then analyzed in either 1% agarose gel (for GFP fragments amplified from the pEGFP-C1 plasmid) pre-stained with either SYBER Gold (Invitrogen), GelRed (Biotium Inc.), or premade 15% NovexTM TBE-Urea Gel (Invitrogen).
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10

TracrRNA Stability Determination in FBS

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To determine the stability of the tracrRNA in vitro, we diluted the tracrRNA to 3 μM, and 1 μL was mixed with 1 μL of 100% FBS in a 10-μL reaction and incubated for the described times at room temperature. The reaction was stopped by adding 2 μL of 20% sodium dodecyl sulfate (SDS) and by loading the RNA onto a 15% Novex TBE-UREA gel (Thermo Fisher Scientific, MA, USA), and was resolved by PAGE. The gel was stained with ethidium bromide and imaged on a Bio-Rad Gel Doc EZ system (Bio-Rad, CA, USA).
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