T4509

Manufactured by Merck Group

Sourced in United States

T4509 is a laboratory equipment product. It is designed for use in scientific research and analysis. The core function of T4509 is to provide a controlled environment for conducting various experiments and tests. Detailed specifications and intended use are not available.

Automatically generated - may contain errors

Lab products found in correlation

12 protocols using t4509

4sU Labeling of iPSCs for RNA-Seq

Check if the same lab product or an alternative is used in the 5 most similar protocols

The experiment should be performed using sufficient cells to generate 25–50 μg of total RNA for each treatment. This is generally equivalent to a 6 or 10 cm dish for most adherent cell lines. At the start of the labeling period, the growth media from the cells is transferred to a sterile 15 ml conical tube, 4sU (Sigma #T4509) is added to a final concentration of 400μM (diluted from a 100 mM stock), and the treated media is put back onto the cells. The published half-life of the GPR56 transcript is 3–4 hours [18 (link)]; therefore, we labeled siRNA-transfected iPSCs with 4sU for 2 hours and 5 minutes. The additional 5 minutes is the estimated time for the cells to take up the 4sU and begin to incorporate it into newly transcribed RNA [26 , 27 (link)].

Russo J., Heck A.M., Wilusz J, & Wilusz C.J. (2017). Metabolic Labeling and Recovery of Nascent RNA to Accurately Quantify mRNA Stability. Methods (San Diego, Calif.), 120, 39-48.

+ Open protocol

+ Expand

4-sU RNA Isolation and Sequencing

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

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.

Audano M., Pedretti S., Ligorio S., Gualdrini F., Polletti S., Russo M., Ghisletti S., Bean C., Crestani M., Caruso D., De Fabiani E, & Mitro N. (2021). Zc3h10 regulates adipogenesis by controlling translation and F-actin/mitochondria interaction. The Journal of Cell Biology, 220(3), e202003173.

+ Open protocol

+ Expand

Transcriptome Profiling of K562 Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

A detailed step-by-step protocol has been deposited in the protocols.io repository (Gressel et al. 2019a ). TT-seq and RNA-seq were performed in 2 biological replicates including RNA spike-ins. Briefly, experiments were performed using

5 \times 10^{7}

K562 cells per biological replicate. Cells were kept at optimal growth conditions and supplemented with 5 µg mL⁻¹ of α-amanitin or solvent (water) for 8 h. After 7 h 55 min, a 4-thiouridine (4sU) labeling pulse (Sigma-Aldrich, T4509) was applied for 5 min using 500 µM (see Supplementary material S3). Total RNA was isolated with the QIAzol reagent (# 79306) according to manufacturer’s instructions except for the addition of 150 ng of RNA spike-in pool with QIAzol reagent as previously described (Schwalb et al. 2016 (link); Gressel et al. 2019a ). The Ovation Universal RNA-Seq System (NuGEN) was used for strand-specific library preparation as described (Gressel et al. 2019b (link)). Purified cDNA libraries were analyzed by Fragment Analyzer prior to Illumina sequencing. Sequencing was performed on a HiSeq 2500 (Illumina) in paired-end mode with 50-bp read length.

Baar T., Dümcke S., Gressel S., Schwalb B., Dilthey A., Cramer P, & Tresch A. (2022). RNA transcription and degradation of Alu retrotransposons depends on sequence features and evolutionary history. G3: Genes|Genomes|Genetics, 12(5), jkac054.

+ Open protocol

+ Expand

eCLIP-qPCR Analysis of RNA-Protein Interactions

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

eCLIP-qPCR was performed as reported before^{41 (link)–43 (link)}. Briefly, cells were cultured in medium with 4-thiouridine (100 μM; T4509, Sigma) for 16 h. Then cells were washed twice by cold PBS and crosslinked with ultraviolet (365 nm,150 mJ/cm²) and then lysed with NP-40 lysis buffer (FNN0021, Invitrogen) containing protease inhibitors and 1 mM dithiothreitol (P2325, Invitrogen). RNase T1 (AM2283, Invitrogen) was added to the supernatant at a final concentration of 1 U/μl and incubated at 22 °C for 15 min. Then Cyclin D1 antibody (RB-010-P, Invitrogen, dilution 1:100) or HA-tag antibody (H9658, Sigma, dilution 1:100) was added and incubated at 4 °C with rotation overnight. Forty microliters of dynabeads Protein G (10003D, Invitrogen) was added and incubated at 4 °C for 3 h. The pellets were incubated in NP-40 lysis buffer with DNase I (18047019, Invitrogen) at a concentration of 1 U/5 μl for 15 min at 37 °C. The immunoprecipitated protein–RNA complex was eluted from the beads by heat denaturing. After SDS-PAGE and nitrocellulose membrane transfer, the 35–110 KD region (a region of 75 kDa (~220 nt of RNA) above the Cyclin D1 protein size) is excised and treated with proteinase K (25530049, Invitrogen) to isolate RNA for next qPCR analysis. Primers were designed every 200 nt with 100-nt overlapping intervals to cover the full length of DILA1, listed in Table S3.

Shi Q., Li Y., Li S., Jin L., Lai H., Wu Y., Cai Z., Zhu M., Li Q., Li Y., Wang J., Liu Y., Wu Z., Song E, & Liu Q. (2020). LncRNA DILA1 inhibits Cyclin D1 degradation and contributes to tamoxifen resistance in breast cancer. Nature Communications, 11, 5513.

+ Open protocol

+ Expand

4sU Labeling of RNA in Transfected and Infected Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For metabolic RNA labelling of 293T transfected with a plasmid or Calu3 infected cells, growth medium was replaced with medium containing 4sU (T4509, Sigma) 24 h post-transfection and 3 hpi at a final concentration of 200 μM (a concentration that did not induce substantial cell cytotoxicity at 3 h labelling). Cells were collected with Tri-reagent at 2, 3, or 4 h after labelling for transfected cells or at 1, 2, or 3 h after labelling for infected cells (corresponding to 4, 5, and 6 hpi). RNA was extracted under reducing conditions and treated with iodoacetamide (A3221, Sigma) as previously described(Herzog et al., 2017 (link)). RNA-seq libraries were prepared and sequenced as described above.

Fisher T., Gluck A., Narayanan K., Kuroda M., Nachshon A., Hsu J.C., Halfmann P.J., Yahalom-Ronen Y., Finkel Y., Schwartz M., Weiss S., Tseng C.T., Israely T., Paran N., Kawaoka Y., Makino S, & Stern-Ginossar N. (2022). Parsing the role of NSP1 in SARS-CoV-2 infection. bioRxiv.

+ Open protocol

+ Expand

Metabolic Labeling and Isolation of Newly Transcribed RNAs

Check if the same lab product or an alternative is used in the 5 most similar protocols

The newly transcribed RNAs were isolated based on metabolic labeling with 4-thiouridine (4sU) as previously described^{52 (link)} with minor modification. Briefly, the cells at 80% confluency were labeled with 400 μM 4sU (T4509, Sigma Aldrich) for 60 min and total RNAs were extracted with TRIzol reagents immediately at the end of labeling period. The next step is biotinylation of total RNA, linking biotin specifically to the thiol group of 4sU labeled transcripts. Biotinylation was conducted by assembling a 500 μl reaction containing 100 μg total RNA, 200 μl Biotin-HPDP (21341, Thermo Fisher Scientific; 1mg/ml in N,N-Dimethylformamide, D4551, Sigma Aldrich), 50 μl 10× Biotinylation buffer (100 mM Tris-HCl pH 7.4, 10 mM EDTA). The reaction was incubated for 1 h at RT in the dark with gentle agitation. The biotinylated RNAs were recovered by isopropanol/ethanol precipitation and resuspended with RNase-free water at approximately 1 μg/μl. To separate the labeled and unlabeled RNA, 100 μg of biotinylated RNAs were mixed with 100 μl μMACS MicroBeads (130–074-101, Miltenyi Biotec) and incubated at room temperature with rotation for 15 min. The labeled RNAs were isolated with μMAC columns according to the instruction, recovered by ethanol precipitation, and subjected to MeRIP seq directly.

Su R., Dong L., Li Y., Gao M., He P.C., Liu W., Wei J., Zhao Z., Gao L., Han L., Deng X., Li C., Prince E., Tan B., Qing Y., Qin X., Shen C., Xue M., Zhou K., Chen Z., Xue J., Li W., Qin H., Wu X., Sun M., Nam Y., Chen C.W., Huang W., Horne D., Rosen S.T., He C, & Chen J. (2022). METTL16 exerts an m6A-independent function to facilitate translation and tumorigenesis. Nature cell biology, 24(2), 205-216.

+ Open protocol

+ Expand

Hypoxia-Induced Transcriptional Dynamics

Check if the same lab product or an alternative is used in the 5 most similar protocols

We used the protocol described by Dolken et al. (2008) and Schwanhausser et al. (2011) (link) that is represented in Figure 1A. Briefly, exponentially growing HUVEC cells were exposed to 21% (normoxia) or 1% (hypoxia) oxygen for 8 h and pulse labeled with 4-thiouridine (400 µM, 4sU, Sigma, T4509) during the last 2 h of treatment. After treatment, total cellular RNA was isolated from cells using TRI-reagent (Ambion, AM9738). An amount of 100 µg of total RNA was subjected to a biotinylation reaction to label the newly transcribed RNA containing the 4sU moiety (Pierce, EZ-Link Biotin-HPDP, 21341). Then, RNA was purified using Ultrapure Phenol:Chloroform:Isoamylalcohol (Invitrogen, 15593 031) and labeled RNA was isolated from the total RNA by affinity chromatography using streptavidin coated magnetic beads (µMacs Streptavidin Kit; Miltenyi, 130074 101).

Tiana M., Acosta-Iborra B., Hernández R., Galiana C., Fernández-Moreno M.Á., Jimenez B, & del Peso L. (2020). Metabolic labeling of RNA uncovers the contribution of transcription and decay rates on hypoxia-induced changes in RNA levels. RNA, 26(8), 1006-1022.

+ Open protocol

+ Expand

4sU Pulsing of HeLa and HEK293T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HeLa or HEK293T Cells were grown to 70–80% confluency and then pulsed by either 50, 100, or 500uM final concentration of 4-Thiouridine (4sU, Sigma T4509) in pre-conditioned cell culture media under standard cell culture conditions. For mitoribosome-IP experiments, one 15 cm plate of cells was used per time point while for all other experiments, one 10 cm plate was used per time point.

McShane E., Couvillion M., Ietswaart R., Prakash G., Smalec B.M., Soto I., Baxter-Koenigs A.R., Choquet K, & Churchman L.S. (2023). A kinetic dichotomy between mitochondrial and nuclear gene expression drives OXPHOS biogenesis. bioRxiv.

+ Open protocol

+ Expand

Metabolic RNA Labeling of Transfected/Infected Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For metabolic RNA labeling of 293T cells transfected with a plasmid or infected Calu3 cells, growth medium was replaced with medium containing 4sU (T4509, Sigma) 24 h post-transfection or 3 hpi at a final concentration of 200 μM (a concentration that did not induce substantial cell cytotoxicity at 3 h labeling). Cells were collected with Tri-reagent at 2, 3, or 4 h after labeling for transfected cells or at 1, 2, or 3 h after labeling for infected cells (corresponding to 4, 5, and 6 hpi). RNA was extracted under reducing conditions and treated with iodoacetamide (A3221, Sigma) as previously described(Herzog et al., 2017 (link)). RNA-seq libraries were prepared and sequenced as described above.

Fisher T., Gluck A., Narayanan K., Kuroda M., Nachshon A., Hsu J.C., Halfmann P.J., Yahalom-Ronen Y., Tamir H., Finkel Y., Schwartz M., Weiss S., Tseng C.T., Israely T., Paran N., Kawaoka Y., Makino S, & Stern-Ginossar N. (2022). Parsing the role of NSP1 in SARS-CoV-2 infection. Cell Reports, 39(11), 110954.

+ Open protocol

+ Expand

Nascent RNA Labeling and Sequencing

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were labeled in media containing 500 μM 4-thiouridine (4sU, Sigma, T4509) for 8 minutes. We found that 8 minutes was the shortest labeling time to obtain enough nascent RNA for nanopore sequencing (>500 ng) from 100 million human cells or 500 million Drosophila cells. K562 cells were harvested in suspension at 0.8–1 million cells/mL. Drosophila S2 cells were labeled as an adherent layer at 95% confluency and lifted with gentle pipetting before harvesting. Cells were collected by centrifugation at 500 g for 2 minutes and washed once in 1X PBS. Samples for total or 4sU labeled RNA were immediately resuspended in Qiazol lysis reagent (Qiagen, 79306) for RNA extraction. Samples for chromatin-associated RNA purification proceeded immediately to the cellular fractionation protocol.

Drexler H.L., Choquet K, & Churchman L.S. (2019). Splicing kinetics and coordination revealed by direct nascent RNA sequencing through nanopores. Molecular cell, 77(5), 985-998.e8.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!