4-sU (Sigma Aldrich, T4509)–labeled RNA isolation was performed as previously described (Rabani et al., 2011 (link)). Briefly, 300 µM 4-sU was added for 10 min to label confluent 36-h differentiated Scramble- and ShZc3h10-treated preadipocytes. Total RNA extraction was performed, as described above, and 1/50 of the total RNA was saved as input. The labeled RNA was isolated and processed as described (Austenaa et al., 2015 (link)). Briefly, the nascent 4-sU–labeled RNA was extracted from 50 µg of total TRIzol-isolated RNA, conjugated to N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (biotin-HPDP; Abcam), and precipitated with 50 µl streptavidin (MyOne Streptavidin T1, Invitrogen). 4-sU RNA samples were eluted in 20 µl of RNase-free water and analyzed by next-generation sequencing. Isolated nascent 4-sU–labeled RNA (20–30 ng) was used for cDNA library synthesis by using the QIAseq Stranded Total RNA Lib Kit (Qiagen, 180745) with no ribosomal depletion or polyA selection. Libraries were sequenced on an Illumina NextSeq 500.
Myone streptavidin t1
Manufactured by Thermo Fisher Scientific
Sourced in Belgium
The MyOne Streptavidin T1 is a magnetic bead-based product designed for the capture and purification of biotinylated molecules. It consists of uniform, superparamagnetic beads coated with streptavidin, a protein with a high affinity for biotin. This product can be used to isolate and enrich various biotinylated targets, such as proteins, nucleic acids, and cells, from complex samples.
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4 protocols using myone streptavidin t1
1
4-sU RNA Isolation and Sequencing
2
Chem-seq: Profiling Protein-DNA Interactions
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As described elsewhere [17 (link)], the Chem-seq procedure was performed as follows: exponentially growing THP 1 cells were fixed with aqueous 1% formaldehyde solution for 20 min in cell culture medium. Chemical crosslinking was terminated, cells were collected and centrifuged, and the derived pellets were washed three times with phosphate buffered saline (PBS). Cell nuclei were prepared using the EZ-Magna Chip Kit (Mercury) as proposed in the kit protocol. Cells were lysed and cell nuclei washed. Nuclei were resuspended and sonicated. Then, magnetic streptavidin dynabeads (MyOne Streptavidin T1, Invitrogen) were preincubated in PBS containing 0.5% bovine serum albumin and either 200 μM biotinylated quercetin or vehicle (DMSO) for 6 h. Polyphenol-bound beads were subsequently washed to remove unbound quercetin. Upon obtaining bead–quercetin complexes, lysates were cleared and incubated with the beads. Beads were washed and bound protein–DNA complexes were eluted. Beads were separated and supernatant was removed to a new tube. Contaminating RNA and protein were digested by addition of RNase and proteinase K, respectively, and the DNA was purified as described in the kit. Finally, purified DNA fragments were quantified by Qbit®.
3
Biotinylated IOX1 enrichment of Th17 cells
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Our method was adopted from a previous study by Anders et al.21 (link) Briefly, biotinylated IOX1 was synthesized by Dangang Peptides Inc. (China). The liquid chromatograph-mass spectrometer and Nuclear Magnetic Resonance were used to verify the product. Total CD4+ T cells were isolated and polarized under the Th17 condition for 3 days. Twenty μM IOX1-biotin or 20 μM biotin was added into the Th17 cells 3 h before harvesting cells. The cells were then fixed with fixation buffer containing 1% formaldehyde at room temperature for 15 min and the fixation was stopped by adding stop buffer for 5 min. After sonication using Bioruptor PLUS (Diagenode, Belgium), all samples were incubated with streptavidin beads (MyOne Streptavidin T1, Invitrogen) in 4 °C overnight, washed with PBS containing 0.1% BSA, and eluted with elution buffer from ChIP kit mentioned above at 70 °C. The immunoprecipitated DNA was then purified and quantified by real-time PCR.
4
Nisin-Inducible Library Screening Protocol
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An aliquot of the NisB library transformed cells
(∼5 × 1010 cells) was inoculated in 50 mL of
GM17EmCm. The culture was grown at 30 °C until an OD600 of about 0.4. At this point, protein and enzyme production was induced
by adding nisin at a final concentration of 5 ng/mL. Incubation was
continued overnight. Library-containing cells were collected by centrifugation,
washed three times with 0.1 M MES-Ca, and resuspended in 2 mL of MES-Ca-T-BSA.
To reduce the number of nonspecifically binding peptides from the
initial library, a 1 mL of library cell suspension was incubated for
30 min at room temperature with 200 μL of biotin-saturated streptavidin-coupled
magnetic beads (Dynabeads and MyOne Streptavidin T1; Invitrogen).
The unbound cells in the supernatant were collected and resuspended
in 1 mL of MES-Ca-T-BSA. For the first round of selection, 200 μL
of streptavidin-coupled magnetic beads was added and incubated under
rotation at room temperature for 1 h. Streptavidin-binding cells were
collected by magnetic-bead assisted cell sorting (MACS), washed 3
times with MES-Ca-T-BSA, resuspended in 50 mL of GM17EmCm, grown overnight
at 30 °C, and used to produce cells for the next round of selection.
Two additional selection rounds were performed. After three rounds
of MACS, the cells were plated, and individual clones were picked
for sequencing analysis.
(∼5 × 1010 cells) was inoculated in 50 mL of
GM17EmCm. The culture was grown at 30 °C until an OD600 of about 0.4. At this point, protein and enzyme production was induced
by adding nisin at a final concentration of 5 ng/mL. Incubation was
continued overnight. Library-containing cells were collected by centrifugation,
washed three times with 0.1 M MES-Ca, and resuspended in 2 mL of MES-Ca-T-BSA.
To reduce the number of nonspecifically binding peptides from the
initial library, a 1 mL of library cell suspension was incubated for
30 min at room temperature with 200 μL of biotin-saturated streptavidin-coupled
magnetic beads (Dynabeads and MyOne Streptavidin T1; Invitrogen).
The unbound cells in the supernatant were collected and resuspended
in 1 mL of MES-Ca-T-BSA. For the first round of selection, 200 μL
of streptavidin-coupled magnetic beads was added and incubated under
rotation at room temperature for 1 h. Streptavidin-binding cells were
collected by magnetic-bead assisted cell sorting (MACS), washed 3
times with MES-Ca-T-BSA, resuspended in 50 mL of GM17EmCm, grown overnight
at 30 °C, and used to produce cells for the next round of selection.
Two additional selection rounds were performed. After three rounds
of MACS, the cells were plated, and individual clones were picked
for sequencing analysis.
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