After pelleting cells at 2880 × g for 5 min, cells were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 500 mM LiCl, 0.5% LiDS, 1 mM EDTA, 5 mM DTT, 1x Protease Inhibitor Cocktail EDTA-free, Roche). Cells were lysed using acid-washed glass beads in a FastPrep device (MPI) using 5 pulses at 6 m/s for 60 sec with 60 sec pausing in between. Lysates were then cleared by centrifugation at 9400 × g for 2.5 min in a table-top centrifuge. Purification of mRNPs was performed using magnetic oligo d(T) beads (NEB) as described in 7 (link), 42 . Finally, cross-links were isolated by C18 chromatography and titanium dioxide solid phase extraction (see below).
Oligo dt magnetic beads
Manufactured by New England Biolabs
Sourced in United States
Oligo(dT) magnetic beads are a laboratory tool used for the purification and isolation of polyadenylated (poly(A)) RNA from biological samples. The beads are coated with oligonucleotides complementary to the poly(A) tail of mRNA, allowing for the selective capture and separation of mRNA molecules from a complex mixture.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Edta free protease inhibitor cocktail, by Roche (2 mentions)
Rnase 1, by Thermo Fisher Scientific (2 mentions)
Benzonase, by Merck Group (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nextflex dna barcode, by PSC Biotech (2 mentions)
Isolate 2 rna kit, by Meridian Bioscience (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Rna library prep kit, by New England Biolabs (2 mentions)
Dnase, by Thermo Fisher Scientific (2 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (2 mentions)
Hiseq platform, by Illumina (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Rnaeasy plus mini kit, by Qiagen (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
30 protocols using oligo dt magnetic beads
1
Isolation of mRNA-binding Protein Complexes
2
Isolation of mRNA-binding Protein Complexes
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After pelleting cells at 2880 × g for 5 min, cells were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 500 mM LiCl, 0.5% LiDS, 1 mM EDTA, 5 mM DTT, 1x Protease Inhibitor Cocktail EDTA-free, Roche). Cells were lysed using acid-washed glass beads in a FastPrep device (MPI) using 5 pulses at 6 m/s for 60 sec with 60 sec pausing in between. Lysates were then cleared by centrifugation at 9400 × g for 2.5 min in a table-top centrifuge. Purification of mRNPs was performed using magnetic oligo d(T) beads (NEB) as described in 7 (link), 42 . Finally, cross-links were isolated by C18 chromatography and titanium dioxide solid phase extraction (see below).
3
Transcriptome Analysis of U-2 OS Cells
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RIC [15 (link)] was performed on nuclear extracts of U-2 OS. Briefly, cells were treated with 10 nM actinomycin D, 2.5 µM CX-5461 or DMSO. Cells were washed with ice-cold PBS, and cross-linked with UVC (150 mJ/cm2) irradiation. Nuclei were isolated as described above and were resuspended in oligo(dT) binding buffer (20 mM Tris pH 7.4, 500 mM LiCl, 0.5% LiDS, 1 mM EDTA, 5 mM DTT). Nuclear lysate was incubated with magnetic oligo(dT) beads (NEB) for one hour at room temperature. Subsequently, the beads were washed and the RNA was eluted from the beads (4). The eluted RNA was supplemented with 2 mM MgCl2, 125U Benzonase (Sigma–Aldrich) and 300U RNase I (Ambion) to digest the RNA.
4
Purification and Elution of mRNPs
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1 × 15 cm dish of cells was used per condition. 2 × binding buffer (BB) (20 mM HEPES-K-NaOH pH 7.5, 1 M NaCl, 1% SDS, 0.2 mM EDTA pH 8) was preheated to 37 °C. 1 × binding buffer was made by mixing 1:1 mRNP lysis buffer (LB) (50 mM HEPES-K-NaOH pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA pH 8.0, 0.5% Igepal Ca-630/NP-40, 0.5% Na-deoxycholate, 10% glycerol) with preheated 2 x BB. 100 µl of magnetic oligo dT beads (New England Biolabs) were used per sample and washed 3 times in 1 × BB. Cells were crosslinked with 300 mJ/cm2 ultraviolet light (UV). Each dish of cells was lysed in 600 µl LB supplemented with protease inhibitor cocktail and RNase inhibitors. Post lysis, samples were spun at 4 °C, 16100 g for 10 minutes. Samples were denatured by adding 1:1 lysate and 2 × BB at 37 °C. Denatured lysates were added to the beads and incubated for 1 hour at 25 °C. Samples were washed 3 times in 1 x BB and eluted in 60 µl of mRNP elution buffer (10 mM Tris pH 7.5, 1 mM EDTA pH 8) supplemented with RNase A (50 µg/mL−1). Elution was carried out at 25 °C shaking at 800 rpm for 30 minutes.
5
mRNP Capture and Elution Protocol
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mRNP capture assay was performed as described previously.53 (link) Cells were UV-crosslinked and scraped from 150 mm dish and lysed in lysis buffer (10 mM Tris pH 7.5, 60 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.2% NP-40, and 1 mM DTT) with complete EDTA-free protease inhibitor cocktail (Roche). Following 10 s sonication, extracts were cleared by centrifugation at 14,000 rpm for 15 min at 4°C. Supernatants were saved, and equal amounts of extracts were incubated with 25 μL of magnetic oligo(dT) beads (New England Biolabs) 1 h at room temperature with rotation. Finally, captured mRNP was eluted from beads with elution buffer (10mM Tris pH 7.5, 1mM EDTA, complete EDTA-free protease inhibitor cocktail and 4 μL of RNAse A/T1 enzyme mix (Thermo Scientific)) for 30 min at 37°C. The elute was precipitated with 20% TCA and the pellet was washed with ice-cold acetone and resus-pended in SDS-PAGE loading buffer for western blotting.
6
Neutrophil RNA-seq protocol from healthy donors
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Neutrophils from 22 healthy donors were isolated using the same approach as for the proteomics samples. RNA was isolated with the RNAeasy plus mini-kit from Qiagen (catalog number 74134) according to the vendor protocol. Magnetic oligo-dT beads (NEB) were used for mRNA enrichment starting with 1 to 10 ng total RNA with RIN values above 9. The NEBNext Ultra II directional RNA Library prep Kit (NEB) was used to prepare RNA-seq libraries according to the manufacturer's protocol. Paired-end sequencing was performed with 2 × 75 cycles on an Illumina NextSeq 500 (Care-for-Rare Genomics Facility at the Dr. von Hauner Children's Hospital). The short reads were aligned with STAR (44 (link)) version 2.5.0a to the human reference genome GRCh37.p13 with basic two-pass method. The counts were then generated using the featureCounts program from the subread toolkit (45 (link)) version 1.5.1. The counts were normalized for sequencing depth and RNA type using DESeq2 (46 (link)). Median transcripts-per-million (TPM) values were calculated by normalizing the median gene read counts to the length of the union of all possible exons coded by a given gene. The raw RNAseq data have been deposited into Gene Expression Omnibus (47 (link)) (GEO) under accession number GSE118644 .
7
RIC-seq Protocol for Studying RNA-Protein Interactions
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RIC was performed on nuclear extracts of MCF10A and U-2 OS. Briefly, cells were treated with 10 nM actinomycin D, 2.5 μM CX5461 or DMSO. Cells were washed with cold PBS, and cross-linked with UVC (150 mJ/cm2) irradiation. Nuclei were isolated as described above and were resuspended in oligo(dT) binding buffer (20 mM Tris pH7.4, 500 mM LiCl, 0.5% LiDS, 1 mM EDTA, 5 mM DTT). Nuclear lysate was incubated with magnetic oligo(dT) beads (NEB) for one hour at room temperature. Subsequently, the beads were washed and the RNA was eluted from the beads (4 (link)). The eluted RNA was supplemented with 2 mM MgCl2, 125U Benzonase (Sigma-Aldrich) and 300U RNase I (Ambion) to digest the RNA.
8
Transcriptome Analysis of Floral Bud Development
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The total RNA was separately extracted and processed from three different development stages of floral buds, including the first development stage (S1), the third development stage (S3), and the fifth development stage (S5) of CH and CL flowers in V. prionantha using an Rneasy Plant Mini Kit (Qiagen, Dusseldorf, Germany). The polyA mRNAs were enriched using the magnetic oligo (dT) beads (NEB, Ipswich, Massachusetts, USA) and then were fragmented into 200 bp pieces. Next, random hexamer (N6) primers (Sangon Biotech, Shanghai, China) were used to build double strand cDNA libraries for all the samples. The cDNA libraries were constructed and sequenced using a BGISEQ-500 platform instrument (BGI, Shenzhen, China). The sequencing reads containing low-quality, adaptor-polluted, and high content of unknown bases (N) were removed before downstream analyses using Trimmomatic (V0.36) [74 (link)] with the parameters of ‘SE -phred33 ILLUMINACLIP: Trimmomatic-0.36/adapters/TruSeq3-SE. fa: 2:30:10 LEADING: 20 TRAILING: 20 SLIDINGWINDOW: 4:20 MINLEN: 50’.
9
Comparative Analysis of Cell Lysis Methods
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Human K562 cells (ATCC) were grown in RPMI medium supplemented with 10% heat-inactivated FBS, 2 mM l -glutamine, 0.1 mM 2-mercaptoethanol, 100 units/ml penicilin G sodium, and 100 μg/ml streptomycin sulfate. NIH3T3 mouse fibroblasts (ATCC) were cultured in DMEM media containing 10% bovine calf serum, 4 mM l -glutamine and 100 units/ml penicillin G sodium and 100 μg/ml streptomycin sulfate. For both K562 and NIH3T3, single cell suspensions of 100 000 cells were prepared and lysed in 500 μl volume either using freeze-thaw cycles (in freeze-thaw lysis buffer; 100 mM Tris, pH 7.5, 10 mM EDTA, 1 M NaCl, 5 μM DTT, 0.4 U/ml Lucigen RNase) or detergent-based buffer as used in DropSeq method (17 (link)) (100 mM Tris, pH 7.5, 10 mM EDTA, 3% Ficoll PM-400, 0.1% Sarkosyl, 25 mM DTT). For freeze-thaw lysis, lysates were prepared for one, two and three freeze-thaw cycles for comparison. Following lysis, mRNA from lysates were isolated using magnetic oligo-dt beads (NEB, S1419S) following manufacturer's instructions. mRNA was quantified using NanoDrop 2000 spectrophotometer. For qPCR, 10 ng of mRNA was used as input for all conditions and reactions were run on CFX Connect Real-Time PCR machine (Biorad) using SsoFast EvaGreen PCR supermix (Biorad) following manufacturer's instructions. Primers were obtained from Sigma (KiCqStart primers).
10
RNA Extraction and Enrichment from Embryos
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For RNA samples, 25 embryos per developmental stage and per replicate were collected and flash frozen in liquid nitrogen. Frozen embryos were thawed and lysed in 1 ml TRIzol (Life Technologies) and total RNA was extracted using the manufacturer’s protocol. Total RNA concentration was calculated by nanodrop.
For polyA-selected RNA samples, polyadenylated RNAs were isolated with oligo(dT) magnetic beads (New England BioLabs, S1419S) according to the manufacturer’s protocol and eluted in 30 µl before nanodrop quantification.
For polyA-selected RNA samples, polyadenylated RNAs were isolated with oligo(dT) magnetic beads (New England BioLabs, S1419S) according to the manufacturer’s protocol and eluted in 30 µl before nanodrop quantification.
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