To extract RNA, a serum sample (50 μL) was added to 200 μL of diethylpyrocarbonate-treated water (Ambion, Carlsbad, CA, USA), which was then added to 750 μL of TRIzol LS Reagent (Ambion). Samples were incubated for 20 min at ambient temperature. After incubation, 200 μL of chloroform (Sigma Aldrich) was added, mixed thoroughly, and incubated for 10 min at ambient temperature. After incubation, samples were centrifuged at 12,000 × g for 15 min at 4°C. A total of 400 μL of the aqueous phase was collected, and the RNA was precipitated by adding 1 μL of GlycoBlue (15 μg/μL) (Ambion) and 400 μL of isopropanol (Sigma Aldrich). Samples were incubated at ambient temperature for 10 min and centrifuged at 12,000 × g for 10 min at 4°C. The resulting pellet was then washed in 1 mL of 75% ethanol (Sigma Aldrich) and centrifuged at 12,000 × g for 5 min at 4°C, after which the pellet was air-dried for 10 min at ambient temperature and resuspended in 50 μL of diethylpyrocarbonate-treated water. Virus RNA was quantified by using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) and primers and a probe specific for the envelope gene (bases 1188–1316) (35 (link)). A standard curve was generated against a synthetic oligonucleotide, and genome copies were expressed as copies per milliliter. The lower limit of detection was 3.0 log10 copies/mL.
Glycoblue
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
GlycoBlue is a nucleic acid co-precipitant and carrier used to facilitate the precipitation and recovery of small amounts of RNA or DNA from solution. It functions by increasing the efficiency of nucleic acid precipitation.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (50 mentions)
Trizol reagent, by Thermo Fisher Scientific (27 mentions)
Proteinase k, by Thermo Fisher Scientific (12 mentions)
Trizol ls, by Thermo Fisher Scientific (10 mentions)
Turbo dnase, by Thermo Fisher Scientific (8 mentions)
Chloroform, by Merck Group (7 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (7 mentions)
Fragment analyzer, by Agilent Technologies (7 mentions)
Isopropanol, by Merck Group (6 mentions)
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (6 mentions)
Bioanalyzer, by Agilent Technologies (6 mentions)
Tbe urea gel, by Thermo Fisher Scientific (6 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (5 mentions)
Rnase free water, by Thermo Fisher Scientific (5 mentions)
Nextseq 500, by Illumina (5 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Promega (4 mentions)
Proteinase k, by Roche (4 mentions)
247 protocols using glycoblue
1
Quantifying Virus RNA from Serum Samples
2
Efficient RNA Extraction from Nuclei and Embryos
3
RNA Immunoprecipitation and Cloning
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Immunoprecipitated RNA (3 μg in 30 μL 10 mM Tris-HCl, pH 7.0) was denatured (2 min, 80°C) and placed on ice. A 5′ adenylated, 3′ blocked with a dideoxy C base cloning linker 5rApp/CTGTAGGCACCATCAAT/3ddC (Integrated DNA Technologies) was ligated to the 3′ ends of RNAs by first dividing the RNA into three microfuge tubes and then adding 10 μL ligation reaction mix to give final concentrations of 50 ng μL−1 cloning linker 1, 12% PEG 8000, 1 × T4 RNA ligase 2 (Rnl2) (truncated) ligation buffer, 10 U μL−1 T4 Rnl2 (truncated) (NEB) and incubating for 3 hr at 37°C. Ligated RNA was incubated (95°C, 35 min) with 20 μL Alkaline fragmentation buffer (100 mM NaCO3 (pH 9.2), 2 mM EDTA) to fragment linker ligated RNA to a narrow size range to reduce size bias of future steps. RNA was precipitated by incubating (30 min, −20°C) with ice cold 500 μL H20, 60 μL 3 M NaOAc (pH 5.5), 2 μL 15 mg mL−1 GlycoBlue (Ambion) and 0.75 mL isopropanol and then spinning (16,000 g, 4°C, 30 min). Pellets were washed with 0.75 mL 80% ethanol, dried (10 min, room temperature) and resuspended sequentially in the same 10 μL 10 mM Tris-HCl (pH 7.0).
4
Efficient RNA Extraction from Nuclei and Embryos
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For RNA extraction, (1) bead-bound nuclei and (2) isolated embryos were immediately incubated with 500 μl of TRIzol reagent (Ambion, CA, USA) for (1) 5 min at RT (vortexing) and (2) 30 min at 60°C, and then purified according to the TRIzol reagent protocol for small sample quantities. GlycoBlue (2 μl of 15 mg/ml; Ambion, TX, USA) was used as a co-precipitant. The RNA was treated with DNase I (~14 Kunitz units; QIAGEN GmbH, Hilden, Germany) for 5 min at RT and then purified (RNeasy Micro Kit; QIAGEN GmbH) and concentrated to 5 μl (Concentrator plus; Eppendorf AG) before being stored at -80°C until further processing.
5
Ribosome Profiling Library Preparation
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Ribosomal footprints were isolated with a sucrose cushion, size-selected, and dephosphorylated as previously described [2 (link), 11 (link)]. Following dephosphorylation of size-selected footprints, we determined the concentration of input material using a Bioanalyzer (RNA 6000 Pico Chip, Agilent Technologies). We found that quantification with a Bioanalyzer was more accurate than with a RNA Qubit or Nanodrop due to the presence of Glycoblue (Ambion) as a precipitant. We used a newly developed kit for small RNA library construction (SMARTer® smRNA-Seq Kit for Illumina®, Clontech catalog number 635030) to generate ligation-free ribosome profiling libraries. Between 1 and 5 ng of size-selected material was used as input and diluted with water to a total volume of 7 μL. Ensuring that reagents remained on ice, polyadenylation mix was prepared by combining 7 μL of RNA input with 2.5 μL of mix 1, which includes poly(A) polymerase. After adding the polyadenylation mix, samples were incubated for 5 minutes at 16 °C. Following incubation, samples were immediately placed on ice to ensure the poly(A) tailing reaction did not continue.
6
Small-RNA Sequencing Library Preparation
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Small-RNA libraries were generated as described in the TruSeq Small RNA Sample Prep Kit manual (Illumina, San Diego, CA, USA), performing 15 cycles in the amplification step. In the purification step after cDNA synthesis, glycoblue (Ambion) was used. The cDNA concentration was measured using the LabChip GX (Caliper). Twenty libraries were pooled equimolarly per lane and sequenced on an Illumina HiSeq2500.
7
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted according to the manufacturer’s instructions using TRI-Reagent. 1 μL Glycoblue (Ambion, Grand Island, NY) was added during the isopropanol precipitation to assist with visualization of the pellet. cDNA synthesis reactions were performed with MMLV Reverse-Transcriptase (Life Technologies, Grand Island, NY), using between 200 ng and 1 μg of total RNA starting material. The cDNA was then diluted in nuclease-free ddH2O to a final concentration of 5 ng/uL of initial RNA concentration to maintain consistency among samples.
8
RNA Immunoprecipitation and Quantification
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RIP was performed as described previously (Jia et al, 2016 ). Briefly, cells were lysed in polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% NP‐40, 1 mM DTT, and 2 mM vanadyl‐ribonucleoside complexes solution [Sigma‐Aldrich, 94742]) supplemented with protease inhibitor cocktail and RNase inhibitor, which were then subjected to IP with indicated antibody, and followed by washing with polysome lysis buffer four times and polysome lysis buffer plus 1 M urea four times. RNAs was released by adding 150 μL of polysome lysis buffer with 0.1% SDS and 45 μg proteinase K (Ambion, AM2548) and incubated at 50°C for 30 min. RNA extracted with phenol–chloroform–isoamyl alcohol mixture was recovered by adding 2 μl GlycoBlue (15 mg/ml, Ambion), 36 μl 3 M sodium acetate, and 750 μl ethanol followed by incubation at −20°C for overnight. Precipitated RNAs were washed with 70% ethanol, air dried, and re‐suspended in RNase‐free water followed by DNase I (Promega, M6101) treatment to remove genomic DNA. The resultant RNAs were subjected to RT‐qPCR analysis.
9
Chromatin Immunoprecipitation (ChIP) in C2C12 Cells
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Transfected C2C12 cells were cross-linked using 1% formaldehyde in PBS for 10 min. Glycine was added to a final concentration of 0.125 M for 5 min, followed by centrifugation. The cell pellet was washed in PBS and subsequently resuspended in ChIP lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% SDS). Chromatin was fragmented using a Covaris sonicator. Before ChIP, equal volumes of ChIP dilution buffer (20 mM Tris-HCl pH 8.0, 1.2 mM EDTA, 1.1% Triton X-100, 200 mM NaCl) was added. Immunoprecipitation was performed using 0.5 mg of chromatin and anti-Flag agarose beads (Sigma) for 2 h at 4 °C. Antibody/protein/DNA complexes were collected, washed and eluted, and cross-links were reversed according to the manufacturer’s instructions. DNA was purified by phenol/chloroform purification using linear acrylamide (Ambion) and GlycoBlue (Ambion) as carriers. ChIP enrichment was analyzed by qPCR using the Mx3000P qPCR System (Stratagene). Primers used for ChIP-qPCR are listed in Supplementary Table 1 .
10
Concentrated RNA extraction from low-input serum
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Due to small volumes and low amounts of RNA in available human serum samples the extraction protocol was adjusted to result in more concentrated RNA. To do this, 50 µl of serum was thawed on ice, mixed with 50 µl of ddH2O and 100 µl of 2× Denaturing Solution (as supplied in the miRVana Paris kit) and kept on ice for 10 min. Samples were spiked with 10 fmoles of a synthetic RNA, Spike1: 5′-UGCUGAAUGCGUAGCUAUAAGC-3′ (IDT) and extracted with an equal volume of acid-phenol chloroform, vortexed for 30 s and centrifuged for 10 min at 10,000 g at RT. The aqueous phase was mixed with 1/10 volume of 3M sodium acetate, 10 µg of GlycoBlue (Ambion) and an equal volume of isopropanol. Samples were allowed to precipitate overnight at −20°C and were then centrifuged at >10,000 g at 4°C. Pellets were washed twice with 75% ethanol, air-dried and then resuspended in 25 µl of 0.1 mM EDTA. The total RNA concentration was below the limit of detection based on Qubit (Invitrogen).
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