Transcriptional responses to SCC1-degradation were profiled by SLAM-seq as described previously (Muhar et al., 2018 (link)) with minor modifications. HeLa SCC1-AID cells were synchronized in G1 by double thymidine block as for PC-HiC. SCC1 degradation was induced by addition of 500 μM auxin (indole-3-acetic acid sodium salt, Sigma-Aldrich). Newly synthesized RNA was labeled by addition of 4-Thiouridine (4SU, Carbosynth) at a final concentration of 100 μM for 60 minutes. Cells were snap-frozen, and extracted total RNA was subjected to alkylation by iodoacetamide (Sigma-Aldrich) for 15 minutes at room temperature in an appropriate buffer. Alkylation was stopped by addition of dithiothreitol (DTT, GE-Healthcare) and alkylated RNA purified by ethanol precipitation. 3′ end mRNA sequencing libraries were generated from 500 ng of alkylated RNA using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen). Single-read sequencing was performed by the Vienna Biocenter Core Facilities (VBCF) for 100 cycles on a HiSeq2500 sequencer (Illumina).
4 thiouridine 4su
Manufactured by Biosynth
Sourced in United Kingdom
4-Thiouridine (4SU) is a synthetic nucleoside analog. It is a derivative of the natural nucleoside uridine, where the oxygen atom at the 4-position of the pyrimidine ring is replaced by a sulfur atom.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500 sequencer, by Illumina (2 mentions)
Dithiothreitol (dtt), by GE Healthcare (2 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Auxin, by Merck Group (1 mentions)
Lps e coli o111 b4, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Flavopiridol, by Merck Group (1 mentions)
Tetracycline hydrochloride, by Merck Group (1 mentions)
4 nitroquinoline n oxide 4 nqo, by Merck Group (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Indole 3 acetic acid sodium salt, by Merck Group (1 mentions)
4 protocols using 4 thiouridine 4su
1
Transcriptional Profiling of SCC1 Degradation
2
Macrophage Transcriptome Labeling with 4sU
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Twenty million primary bone marrow-derived macrophages were plated on 15-cm cell culture dishes in serum-free macrophage medium. Cells were pre-treated with either 1 μM Dexamethasone (Dex, Sigma-Aldrich, Cat. No.: D4902) or vehicle (Ethanol) for 30 min, followed by 2 h of 100 ng/mL LPS treatment (LPS E. COLI O111:B4, Sigma-Aldrich, Cat. No.: LPS25). After 1 h of LPS treatment, 200 μM of 4-thiouridine (4sU, Carbosynth Ltd., UK, Cat. No.: 13957-31-8) was added to label nascent transcripts. Note, the 4-thiouridine concentration and incubation time were optimized concerning cell viability and incorporation efficiency as determined by dot plot assays (data not shown). All treatments were performed in dark humidified incubators at 37 °C and 5% CO2. After the treatment, the cell culture medium was removed, and cells were washed in D-PBS and collected in 10 mL Trizol (Invitrogen, Waltham, MA, USA) per 15-cm dish.
3
Preparation and Storage of Bioactive Compounds
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4-Nitroquinoline N-oxide (4-NQO; Sigma) was diluted in DMSO to a final concentration of 50 mM, aliquoted, sealed, and stored at −80°C. UV irradiation was performed in Crosslinker CL-1000 using 254 nm wavelength lamp with dose 40-60 J/m2. Flavopiridol (Sigma) and 4-Thiouridine (4sU; Carbosynth) were diluted in DMSO to a final concentration of 1 mM and stored at −20°C. Tetracycline hydrochloride (Sigma) was diluted in water to a final concentration of 1 mg/ml and stored at −20°C. SB203580 (Selleckchem) was diluted in DMSO to a final concentration of 50 mM and stored in −20°C.
4
Transcriptional Responses to SCC1 Degradation
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Transcriptional responses to SCC1-degradation were profiled by SLAM-seq as described previously (Muhar et al., 2018) with minor modifications. HeLa SCC1-AID cells were synchronized in G1 by double thymidine block as for PC-HiC. SCC1 degradation was (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted February 12, 2020. ; https://doi.org/10.1101/2020.02.10.941989 doi: bioRxiv preprint induced by addition of 500 µM auxin (indole-3-acetic acid sodium salt, Sigma-Aldrich). Newly synthesized RNA was labelled by addition of 4-Thiouridine (4SU, Carbosynth) at a final concentration of 100 µM for 60 minutes. Cells were snap-frozen and extracted total RNA was subjected to alkylation by iodoacetamide (Sigma-Aldrich) for 15 minutes at room temperature in an appropriate buffer. Alkylation was stopped by addition of dithiothreitol (DTT, GE-Healthcare) and alkylated RNA purified by ethanol precipitation. 3' end mRNA sequencing libraries were generated from 500 ng of alkylated RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen). Single-read sequencing was performed by the Vienna Biocenter Core Facilities (VBCF) for 100 cycles on a HiSeq2500 sequencer (Illumina).
The copyright holder for this preprint this version posted February 12, 2020. ; https://doi.org/10.1101/2020.02.10.941989 doi: bioRxiv preprint induced by addition of 500 µM auxin (indole-3-acetic acid sodium salt, Sigma-Aldrich). Newly synthesized RNA was labelled by addition of 4-Thiouridine (4SU, Carbosynth) at a final concentration of 100 µM for 60 minutes. Cells were snap-frozen and extracted total RNA was subjected to alkylation by iodoacetamide (Sigma-Aldrich) for 15 minutes at room temperature in an appropriate buffer. Alkylation was stopped by addition of dithiothreitol (DTT, GE-Healthcare) and alkylated RNA purified by ethanol precipitation. 3' end mRNA sequencing libraries were generated from 500 ng of alkylated RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen). Single-read sequencing was performed by the Vienna Biocenter Core Facilities (VBCF) for 100 cycles on a HiSeq2500 sequencer (Illumina).
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