Total RNA (300 μg) extracted from J82 cells was added to an appropriate buffer with 5′-biotinylated PNA oligomer uc.8+ (100 pmol) and was incubated overnight at 4°C with rotation. After complex formation, μMACS Streptavidin MicroBeads (100 μL, Miltenyi Biotec) were added to the mixture and incubated for 30 min at 4°C. μMACS columns were equilibrated with equilibration buffer for nucleic acids (100 μL) and were rinsed with the same buffer used for the binding reaction. Labeled complexes were applied to the top of the column matrix. Columns were washed with washing buffer (4 × 100 μL) to remove molecules with nonspecific binding. RNA labeled with biotinylated PNA oligomer was eluted with elution buffer (150 μL, μMACS Streptavidin kit 130-074-101, Miltenyi Biotec) according to the manufacturer's instructions. We used a 5′-biotinylated scrambled oligonucleotide as a control.
μmacs streptavidin microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
μMACS Streptavidin MicroBeads are superparamagnetic beads coated with streptavidin, a protein that has a high affinity for biotin. These beads are designed for the isolation and separation of biotinylated molecules, cells, or other biological components from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Infinite m1000 pro plate reader, by Tecan (2 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (2 mentions)
μcolumn, by Miltenyi Biotec (2 mentions)
Minielute column, by Zymo Research (2 mentions)
Epiquik m6a rna methylation quantification kit, by Epigentek (2 mentions)
Biotin 16 utp, by Roche (1 mentions)
Zymo clean concentrator 5 kit, by Zymo Research (1 mentions)
Ez link hpdp biotin, by Thermo Fisher Scientific (1 mentions)
Truseq rna sample prep v2 ls protocol, by Illumina (1 mentions)
Minelute spin column, by Qiagen (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
7 protocols using μmacs streptavidin microbeads
1
Affinity Purification of Biotinylated RNA
2
Biotin-labeled RNA Purification and Isolation
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Transcription reactions with pRST5 DNA were carried out for 30 min at 37°C using ribonucleotides at 10 mM concentration including a mixture of biotin-16-UTP (Roche Diagnostics GmbH) and unmodified UTP at a molar ratio of 1:4. The samples were then treated with RNase A and H for 1 h at 37°C followed by deproteinization with Proteinase K and SDS at 37°C. The samples were filtered through agarose beads and the peak fractions pooled. Paramagnetic iron particles (μMACS streptavidin microbeads, Miltenyi Inc.) (3 μl) were added to a 50 μl aliquot of the purified t-loop molecules and allowed to incubate overnight at 4°C. The samples were then filtered through agarose beads to remove the unbound beads and prepared for EM.
3
Biotinylation and Purification of RNA
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100 µg RNA was biotinylated using MTSEA biotin-XX linker (Biotinum, Cat.# 90066) and purified by μMACS streptavidin MicroBeads (Miltenyi Biotec, 130-092-948) as described by Gregersen et al.60 (link). with the following modifications. The µColumn (Miltenyi Biotec, 130-092-948) was washed three times with 1 ml of 65 °C pre-warmed pull-out wash buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% TWEEN20 (v/v)), followed by three washes with 1 ml pull-out wash buffer at room temperature. The eluted RNA was purified using the ZYMO Clean & Concentrator-5 kit (ZYMO Research, Cat.# R1013).
4
Enrichment of Biotinylated RNA Targets
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Total RNA (300 μg) extracted from J82 cell line was incubated in an appropriate buffer with 100 pmol of 5′-biotinylated oligonucleotides T-UCR 8+, T-UCR 201+, T-UCR 128+ and one oligonucleotide scramble in miR-596 binding site overnight at 4 °C with rotation. After complex formation, 100 μL of μMACS Streptavidin MicroBeads (Miltenyi Biotec, Bergisch, Gladbach, Germany) were added and incubated for 30 min at 4 °C. μMACS columns were equilibrated with 100 μL of Equilibration buffer (for nucleic acid; supplied with the kit) and rinsed with the same buffer used for the binding reaction. Labelled complex was applied onto the top of the column matrix. Columns were washed with 4 × 100 μL of washing buffer (supplied with the kit) to remove non-specifically binding molecules. RNA labelled to biotinylated oligonucleotides was eluted with 150 μL of elution buffer (supplied with the kit) according to manufacturer’s instructions μMACSTM Streptavidin Kit).
5
Quantification of m6A RNA Methylation
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Total RNA from E. coli Tuner (DE3) cells was purified as described above and adjusted to a volume of 200 μL. Total RNA was denatured at 75 °C for 5 min and incubated at 42 °C for 10 min with 1 μg of dual biotin labeled DNA probe complimentary to a unique 30 base region within the synthetic target. Probe annealed target RNA was bound to 100 μL of μMACS Streptavidin microbeads (Miltenyi Biotec) and incubated at 4 °C for 15 min. Magnetic separation was done with a μColumn (Miltenyi Biotec). RNA:probe was passed over the μColumn and washed twice with 150 uL TE buffer. Probe bound RNA was eluted by adding 150 μL of H2O warmed to 90 °C. Eluted RNA was purified and concentrated using a MiniElute column (Zymo Research). Resulting RNA concentration was determined using a Qubit RNA HS assay kit (Thermo Fisher). RNA was serial diluted and m6A was quantified with the EpiQuik m6A RNA methylation quantification kit (Epigentek) according to manufacturer’s protocol. Flourometric m6A signal was measured on an Infinite Pro M1000 plate reader (Tecan) at 530EX/590EM nm.
6
Quantification of m6A RNA Methylation
7
Measuring Global mRNA Half-Lives in Cell Lines
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For global mRNA half-life measurements, MCF7 and HEK293 cells were labeled with 700 μM 4SU for 60 minutes. Total RNA was extracted using the miRNeasy kit (QIAGEN). 4SU residues were biotinylated using EZ-Link biotin-HPDP (Thermo Fisher Scientific (Waltham, MA, USA)). Biotinylated 4SU-labled RNA was separated from non-labeled RNA using μMACS Streptavidin MicroBeads (Miltenyi (Bergisch Gladbach, Germany)) and 4SU-labeled RNA was eluted from μColumns by addition of 100 mM DTT. RNA was recovered from the flow-though and 4SU-labeled fractions using MinElute Spin columns (QIAGEN). Input (total), flow-though (non-labeled RNA) and eluted (4SU-labled RNA) samples were used for poly(A) + mRNA library preparation following the TruSeq RNA sample Prep v2 LS protocol (Illumina). The libraries were sequenced on an Illumina Hiseq 2500 for 100 cycles (multiplexed 1 × 101 + 7 index). mRNA half-lives were computed from gene-wise FPKM (fragments per kilobase of exonic sequence per million fragments mapped) as previously described [51 (link)]. To access changes in mRNA half-life, we computed the log2 fold change of all measured genes on quantile normalized data.
MCF7 and HEK293 half-life measurement sequencing data have been deposited under GEO accession number GSE49831.
MCF7 and HEK293 half-life measurement sequencing data have been deposited under GEO accession number GSE49831.
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