Primary transcripts were enriched following a protocol adapted from a previously published method8 (link). 5 μg of total RNA was mixed with 5 μl of 10× VCE Buffer (New England BioLabs, M2080) in a total volume of 50 μl, incubated for 2 min at 70 °C, and then placed on ice. 5 μl of 3’-Desthiobiotin-GTP (New England BioLabs, N0761) and 5 μl of Vaccinia virus Capping Enzyme (New England BioLabs, M2080) were added to the reaction and incubated at 37 °C for 30 min. After purification with 1.5× RNAClean beads, the capped RNA was eluted and subjected to 3’ adaptor ligation as described above. The RNA was cleaned twice with 1.5× RNAClean beads and then enriched with Hydrophilic Streptavidin Magnetic Beads (New England BioLabs, S1421). After washing thoroughly four times with Binding Buffer (10 mM Tris‐HCl pH 7.5, 2 M NaCl, 1 mM EDTA) and three times with Washing Buffer (10 mM Tris‐HCl pH 7.5, 0.25 M NaCl, 1 mM EDTA), the RNA was eluted with 26 μl of Biotin Buffer (10 mM Tris‐HCl pH 7.5, 0.5 M NaCl, 1 mM EDTA, 1 M biotin) and incubated at 37 °C for 25 min on a rotator. Then 14 μl of Binding Buffer was added and incubated for another 4 min. The RNA was cleaned with 1.5× RNAClean beads and eluted in 12 μl of H2O. The 5’ capped and 3’ ligated RNA was reverse transcribed by the maturase as described above.
Hydrophilic streptavidin magnetic beads
Manufactured by New England Biolabs
Sourced in United States
Hydrophilic Streptavidin Magnetic Beads are a type of magnetic bead coated with streptavidin, a protein that binds strongly to biotin. These beads are hydrophilic, meaning they have a high affinity for water molecules. The magnetic properties of the beads allow for easy separation and manipulation using a magnetic field.
Automatically generated - may contain errors
Lab products found in correlation
3 desthiobiotin gtp, by New England Biolabs (3 mentions)
Vaccinia virus capping enzyme, by New England Biolabs (2 mentions)
Vce buffer, by New England Biolabs (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Polarstar omega plate reader, by BMG Labtech (1 mentions)
Qubit 3, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer dna 1000, by Agilent Technologies (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Rna clean and concentrator kit, by Zymo Research (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Vaccinia capping system, by New England Biolabs (1 mentions)
Accel ngs methyl seq dna library kit, by Integrated DNA Technologies (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Dykddddk tag monoclonal antibody fg4r, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
9 protocols using hydrophilic streptavidin magnetic beads
1
Primary Transcript Enrichment Protocol
2
Primary Transcript Enrichment Protocol
3
Quantifying NCAM2 Peptide Cleavage
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Hippocampal tissue or synaptosomes containing 1 mg of total protein were lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na4P2O7, 1 mM NaF, 1% (v/v) Triton X-100, protease inhibitor cocktail (set III, Merck)) for 30 min at room temperature. Lysates were centrifuged for 15 min at 20,000g at 4 °C. Peptides corresponding to amino acids 682–701 (NCAM2aa682-701) and 666-685 (NCAM2aa666-685) of human NCAM2, and mutated NCAM2aa682-701 peptides with aspartic acid 693 exchanged to alanine or asparagine 689 exchanged to alanine were purchased from Peptide 2.0 (Chantilly, VA, USA). The peptides contained FITC and biotin at the N- and C-termini, respectively. Peptides (250 ng ml−1 final concentration) were mixed with lysates of hippocampal tissue or synaptosomes (20 μg of total protein in 1 ml of TBS) and incubated for 1 h at room temperature. Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature. Fluorescence of FITC groups attached to fragments of cleaved peptides remaining in the solution was measured using POLARstar Omega plate reader (BMG Labtech).
4
Cappable-seq Library Generation from RNA
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RNA samples from triplicate normoxic and hypoxic cultures were subjected to enrichment for primary transcripts as previously described [21 (link)], with minor modifications. First, 1.8× Agencourt AMPure XP beads (Beckman) were used to purify RNA in all procedures. Second, to obtain enough primary transcripts for subsequent library construction, enrichment procedure using hydrophilic streptavidin magnetic beads (NEB) was performed for one time. The NEBNext Small RNA Library Prep Set for Illumina (NEB) was used to generate Cappable-seq libraries. To reduce the concentration of adaptor dimer, the 3′ SR adaptor and 5′ SR adaptor were both used at 4-fold dilutions. After 21 cycles of PCR amplification, the libraries were purified using a QIAquick PCR Purification Kit (QIAGEN) and 1.5× Agencourt AMPure XP beads (Beckman). The concentration and size distribution of the libraries were determined by Qubit 3 (Invitrogen) and Bioanalyzer DNA 1000 (Agilent) respectively. Sequencing was performed by the Illumina HiSeq X Ten platform.
5
Isolation and Purification of Capped RNA
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50 μL of eluted RNA was mixed with 50 μL of 10 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA (wash Buffer A). This mix was added to 100 μL of hydrophilic streptavidin magnetic beads (New England Biolabs) previously washed twice with wash Buffer B (10 mM Tris-HCl, pH 7.5, 50 mM NaCl) and twice with wash Buffer A (100 μL/wash). The RNA-bead mixture was incubated for 20 min at room temperature with occasional resuspension. Using a magnetic rack, the RNA-bead mixture was washed two times with wash Buffer A, followed by two washes with wash Buffer B (100 μL/wash). All washes (containing unlabeled RNA) were collected, cleaned up using the RNeasy Kit (Qiagen) and stored at −80°C until further use (‘Uncapped RNA’ sample). Beads (containing 3´ desthiobiotin capped RNA) were resuspended in wash Buffer B containing 1 mM biotin and incubated for 20 min at room temperature with occasional mixing. After placing the tube in the magnetic rack again, the supernatant (100 μL), containing the 3´ desthiobiotin capped transcripts was collected and cleaned up using the RNA Clean and Concentrator Kit™ (Zymo), and the RNA eluted in 10 μL H2O and stored at −80°C until further use (‘Capped RNA’ sample).
6
Primary Transcript Enrichment for RNA-seq
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A 5 μg quantity of total RNA was used for primary transcript enrichment with our previously published method3 (link). In brief, the 5′-triphosphorylated RNA species was specifically capped with 3′-desthiobiotin-GTP (New England BioLabs, N0761) by the Vaccinia Capping System (New England BioLabs, M2080S). The RNA was subjected to 3′ adaptor ligation using the same procedure as described above and subsequently enriched with Hydrophilic Streptavidin Magnetic Beads (New England BioLabs, S1421). After washing thoroughly, the RNA was eluted and reverse transcribed to cDNA as described above. The remaining steps were the same as those for library preparation for total RNA SEnd-seq, except that the DNA library was amplified for 15 cycles.
7
EMCV Genomic Sequence Enrichment and Sequencing
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The cytoplasmic fractions were separated and nucleic acids were extracted as described above. The customized panel of probes that cover the full-length antisense EMCV genomic sequence was obtained from iGeneTech Bioscience (Beijing, China). The extracted nucleic acids were then hybridized with the probes using the TargetSeq enrichment kit (iGeneTech Bioscience, 324091-V3) according to the manufacturer’s instructions. Hybridized vDNA was captured by Hydrophilic Streptavidin Magnetic Beads (New England Biolabs, S1421) and then eluted with nuclease-free water on a magnetic separator immediately after heating at 98 °C for 5 min. The enriched vDNA was fragmented by a brief sonication. The ssDNA libraries were prepared using the Accel-NGS Methyl-Seq DNA Library kit (Swift Biosciences, 30024) following the manufacturer’s instructions. The libraries were sequenced on Illumina Novaseq 6000 using the paired-end sequencing strategy. The DNA library preparation, high-throughput sequencing and data analysis were conducted by Seqhealth Technology Co., Ltd (Wuhan, China).
8
ChIP-seq Protocol for dCas9 Protein
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Cells expressing gDNA and in control (transfected with GAL4 plasmid) groups were all expanded to 150 mm culture dishes, crosslinked with 2% formaldehyde, and quenched with 0.25 M glycine16 (link). Cells were treated with cell lysis buffer and nuclear lysis buffer to isolate chromatin. Then the separated chromatin was washed with the mixture of 8 M urea, re-suspended in the IP-binding buffer, and sonicated into ~500 bp length of segments. Hydrophilic streptavidin magnetic beads (NEB, Ipswich, MA, USA) were added into the IP-binding buffer and rotated overnight at 4 °C to isolate dCas9 protein. Isolated proteins were subjected to western blot analysis and SDS-PAGE. Proteins in SDS-PAGE gels were visualized after silver staining15 (link),16 (link).
9
Pol II Transcription Assay with Curaxins
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The competitor core nucleosomes were assembled using 5′ end-biotinylated 147-bp DNA template. Transcription by Pol II in the presence or absence of curaxins, FACT, and competitor core nucleosomes (containing 50% of end-biotinylated DNA) was conducted as described above. After transcription, hydrophilic streptavidin magnetic beads (NEB) were added to the reaction mixture for 10 min at room temperature, and the supernatant was collected. The beads were resuspended in 1× TB40 buffer. The SDS loading buffer [1× SDS loading buffer containing 50 mM tris-HCl (pH 6.8), 2% SDS, 70 mM β-mercaptoethanol, and 10% glycerol] was added to the samples and heated at 99°C for 10 min. Electrophoresis and blotting were done using NuPAGE (4 to 12% bis-tris) (Invitrogen), NuPAGE MOPS SDS Running Buffer (Invitrogen), PVDF membrane (Invitrogen), and film developing solutions (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific). The Flag-tagged SPT16 protein was detected using the DYKDDDDK Tag Monoclonal Antibody (FG4R) (Invitrogen). The data were quantified using OptiQuant software and normalized for total protein loading.
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