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Stratalinker

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The Stratalinker is a laboratory instrument designed for UV cross-linking. It provides controlled exposure of samples to ultraviolet light, which can be used for various applications such as nucleic acid or protein immobilization.

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Lab products found in correlation

Fragmentation reagent, by Thermo Fisher Scientific (2 mentions) Magnetic stand, by Thermo Fisher Scientific (2 mentions) Ribominus, by Thermo Fisher Scientific (2 mentions) Protein a g bead, by Thermo Fisher Scientific (2 mentions) Dynabeads oligo dt, by Thermo Fisher Scientific (1 mentions) Tth buffer, by Promega (1 mentions) T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)

7 protocols using stratalinker

1

Profiling RNA-Protein Interactions with CRAC

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UV crosslinking and analysis of cDNA (CRAC) experiments were performed as previously described (34 (link),48 (link)). For PAR-CRAC, cells were grown in media supplemented with 100 μM 4-thiouridine for 6 h prior to crosslinking at 365 nm in a Stratalinker (Agilent) using 2 cycles of 180 mJ/cm2. Samples were then processed as for UV-CRAC and the obtained sequencing data were analysed as previously described (49 (link)) with the exception that for PAR-CRAC samples, only sequence reads containing T-to-C mutations were mapped to the human genome.
Memet I., Doebele C., Sloan K.E, & Bohnsack M.T. (2017). The G-patch protein NF-κB-repressing factor mediates the recruitment of the exonuclease XRN2 and activation of the RNA helicase DHX15 in human ribosome biogenesis. Nucleic Acids Research, 45(9), 5359-5374.
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2

Detecting m6A RNA Modifications by miCLIP

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For initial miCLIP libraries, total RNA was purified from HEK293 cells. Poly(A)+ RNA was prepared from four biological replicates of HEK293 cells and processed in parallel for miCLIP. Mouse liver RNA was depleted of ribosomal RNA by Ribominus (Life Technologies).
RNA was fragmented using fragmentation reagent (Life Technologies) to a size between 30 and 130 nt. After stopping the reaction, 20 μg fragmented RNA was directly diluted in 450μl IP buffer (50 mM Tris pH 7.4, 100 mM NaCl, 0.05% NP40) and incubated with 1–5 μg anti-m6A antibody for 1–2 h at 4°C rotating head over tail. The solution was then transferred into a 3cm cell culture dish and crosslinked twice with 0.15 J cm−2 UV light (254 nm) in a Stratalinker (Agilent).
After crosslinking, the solution was transferred into Eppendorf tubes and incubated with 30 μl Protein A/G beads (Thermo Scientific) for 1–2 h at 4°C, rotating. Bead-bound antibody-RNA complexes were then recovered on a magnetic stand (Life technologies) and washed twice with high-salt buffer (50 mM Tris pH7.4, 1M NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS), twice with IP buffer, and twice with PNK wash buffer (20 mM Tris, 10 mM MgCl2, 0.2% Tween20).
Linder B., Grozhik A.V., Olarerin-George A.O., Meydan C., Mason C.E, & Jaffrey S.R. (2015). Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome. Nature methods, 12(8), 767-772.
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3

Detecting m6A RNA Modifications by miCLIP

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For initial miCLIP libraries, total RNA was purified from HEK293 cells. Poly(A)+ RNA was prepared from four biological replicates of HEK293 cells and processed in parallel for miCLIP. Mouse liver RNA was depleted of ribosomal RNA by Ribominus (Life Technologies).
RNA was fragmented using fragmentation reagent (Life Technologies) to a size between 30 and 130 nt. After stopping the reaction, 20 μg fragmented RNA was directly diluted in 450μl IP buffer (50 mM Tris pH 7.4, 100 mM NaCl, 0.05% NP40) and incubated with 1–5 μg anti-m6A antibody for 1–2 h at 4°C rotating head over tail. The solution was then transferred into a 3cm cell culture dish and crosslinked twice with 0.15 J cm−2 UV light (254 nm) in a Stratalinker (Agilent).
After crosslinking, the solution was transferred into Eppendorf tubes and incubated with 30 μl Protein A/G beads (Thermo Scientific) for 1–2 h at 4°C, rotating. Bead-bound antibody-RNA complexes were then recovered on a magnetic stand (Life technologies) and washed twice with high-salt buffer (50 mM Tris pH7.4, 1M NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS), twice with IP buffer, and twice with PNK wash buffer (20 mM Tris, 10 mM MgCl2, 0.2% Tween20).
Linder B., Grozhik A.V., Olarerin-George A.O., Meydan C., Mason C.E, & Jaffrey S.R. (2015). Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome. Nature methods, 12(8), 767-772.
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4

Site-Specific Detection of m6A in mRNA

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For site-specific detection of m6A, DNA primer (5′-TGCTTAACTCGAAGGTATCTTTG -3′) was firstly 5′ labeled with 32P using T4 polynucleotide kinase (Invitrogen) and [γ-32P] ATP (Perkin Elmer) and purified by ethanol precipitation. The Poly(A)+ RNA was prepared using Dynabeads® Oligo (dT) (Thermo Fisher Scientific) from MEF cells under normal or amino acid starvation condition. 10 μg mRNA was directly diluted in 450 μl immunoprecipitation buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.05% NP-40) and incubated with 2.5 μg m6A antibody at 4 °C for 3 h, rotating head over tail. The solution was then cross-linked twice with 0.15 J cm−2 UV light (254 nm) in a Stratalinker (Agilent). After cross-linking, the reverse transcription was conducted with indicated 32P-labeled primer and Tth enzyme as previous described. Generally, 10 μg m6A-crosslinked mRNA was prepared in a total volume of 6 μL with Tth buffer (Promega) and 1 μL radiolabeled primer. The mixture was heated at 95°C for 10 min and cooled slowly to room temperature. The annealing solution were combined with 5 U of Tth enzyme, 1 mM MnCl2 and heated at 55°C for 3 min. After adding the dTTP solution (final dTTP concentration: 100 μM), the reactions were heated for 10 minutes at 55°C. Reaction products were resolved on a 20% denaturing polyacrylamide gel and detected by autoradiography overnight.
Zhou J., Wan J., Shu X.E., Mao Y., Liu X.M., Yuan X., Zhang X., Hess M.E., Brüning J.C, & Qian S.B. (2018). N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response. Molecular cell, 69(4), 636-647.e7.
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5

Quantifying XPC Enrichment at CPD Lesions

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Cells were grown on coverslips (Fisher Scientific). Before irradiation, the coverslips were covered by 5 µm nanopore filters (Millipore) to allow local UV-C irradiation. The cells were then irradiated with 100 J/m2 UV-C light (Stratalinker, Agilent Genomics) and fixed 10 min after irradiation. The immunofluorescence was essentially done as described above, with the addition of a denaturation step with 0.07 M NaOH in PBS for 5 min and a second blocking step in PBS + before incubation with the primary antibody, to allow recognition of CPD lesions. The enrichment of XPC at CPD lesions was quantified in ImageJ. Nuclei and CPD lesions were recognized by thresholding the Hoechst and CPD signal, respectively. The mean fluorescent intensity of XPC and CPD was measured at CPD spots and in the rest of the nucleus. The enrichment of XPC/CPD at UV-C lesions was quantified as followed: mean fluorescence(spot)/mean fluorescence(nucleus background)−1.
Blessing C., Apelt K., van den Heuvel D., Gonzalez-Leal C., Rother M.B., van der Woude M., González-Prieto R., Yifrach A., Parnas A., Shah R.G., Kuo T.T., Boer D.E., Cai J., Kragten A., Kim H.S., Schärer O.D., Vertegaal A.C., Shah G.M., Adar S., Lans H., van Attikum H., Ladurner A.G, & Luijsterburg M.S. (2022). XPC–PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair. Nature Communications, 13, 4762.
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6

Identification of Photoreactive Protein Targets

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Cells were incubated for 30 minutes in 10 nM or 100 nM CRT0105481 then exposed to 120 mJ 254 nm UV light using a Stratalinker (Agilent, Santa Clara, CA) and harvested for 2D gel analysis and autoradiography. Gels were stained with Simply Blue Safe Stain (Thermo Fisher Scientific, York, UK), infused with En3hance autoradiography enhancer reagent (Perkin Elmer, Warrington, UK), dried, and incubated with radiographic film for 1 week (100 nM CRT0105481 gels) or 1 month (10 nM CRT0105481 gels) at −70 °C. Labeled spots were excised for LC–MS/MS analysis. Recombinant human proteins PGK1 (ab211320) and DJ1 (ab51198) for in vitro labelling were obtained from Abcam (Cambridge, UK).
Bilsland A.E., Liu Y., Turnbull A., Sumpton D., Stevenson K., Cairney C.J., Boyd S.M., Roffey J., Jenkinson D, & Keith W.N. (2019). A Novel Pyrazolopyrimidine Ligand of Human PGK1 and Stress Sensor DJ1 Modulates the Shelterin Complex and Telomere Length Regulation. Neoplasia (New York, N.Y.), 21(9), 893-907.
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7

DHX37 RNA-Protein Interactions Mapping

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CRAC was performed as previously described in references 38,40,51,52. Briefly, expression of DHX37-Flag or the Flag tag was induced in HEK293 cell lines for 24 h before growth in media supplemented with 100 μM 4-thiouridine for 6 h. In vivo crosslinking was then performed using 2 cycles of 180 mJ/cm2 light at 365 nm in a Stratalinker (Agilent Technologies). Cells were harvested and RNA-protein complexes were enriched by tandem affinity purification on anti-Flag M2 magnetic beads and NiNTA. After a limited RNase digestion, co-precipitated RNAs were 5ʹ end labelled with [32P] and sequencing adaptors were ligated. RNA-protein complexes were separated by denaturing PAGE, transferred to a nitrocellulose membrane and labelled RNAs were visualized by autoradiography. Regions of the membrane were excised and RNAs were eluted by Proteinase K digestion then extracted using PCI. RNA fragments were reverse transcribed, amplified by PCR and the resulting library was subjected to Illumina sequencing. The obtained sequence reads were mapped to the human genome (GRCh37.p13).
Choudhury P., Hackert P., Memet I., Sloan K.E, & Bohnsack M.T. (2018). The human RNA helicase DHX37 is required for release of the U3 snoRNP from pre-ribosomal particles. RNA Biology, 16(1), 54-68.
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