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Cap clip

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Cap-Clip is a lab equipment product that securely attaches caps to microcentrifuge tubes. It provides a simple and effective way to keep caps in place during various laboratory procedures.

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Lab products found in correlation

Magnetic mrna isolation kit, by New England Biolabs (3 mentions) Anti m6a antibody, by Abcam (2 mentions) Novaseq 6000, by Illumina (2 mentions) Fragmentation reagents kit, by Thermo Fisher Scientific (2 mentions) T4 pnk, by New England Biolabs (2 mentions) Nuclease p1, by Merck Group (2 mentions) Alkaline phosphatase, by Merck Group (2 mentions) Lightcycler 480, by Roche (1 mentions) Truseq mrna library preparation kit, by Illumina (1 mentions) Phusion high fidelity dna polymerase, by Illumina (1 mentions) Dynabeads, by Illumina (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Novaseq 6000 platform, by Illumina (1 mentions) Dynabead, by Thermo Fisher Scientific (1 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) T4 rna ligase 1, by New England Biolabs (1 mentions) Dnase, by New England Biolabs (1 mentions) Ammonium bicarbonate, by Merck Group (1 mentions) Tri reagent, by Merck Group (1 mentions)

Spelling variants of this product

Cap-Clip Acid Pyrophosphatase (15 citations) Cap-Clip enzyme (3 citations)

6 protocols using cap clip

1

m6Am-exo-seq and qPCR for Epitranscriptomic Analysis

Cited 1 time
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m6Am-exo-seq was performed according to protocols developed by the Shi group (24 (link)). Briefly, mRNA was extracted from PCIF1-KO and control Calu-3 cells using a Magnetic mRNA Isolation Kit. Aliquots (100 μg) of mRNA were fragmented using a Fragmentation Reagents Kit (Invitrogen, AM8740) according to the manufacturer’s protocol. Fragmented mRNAs were phosphorylated with T4 PNK (NEB, M0201S) and then dephosphorylated with Terminator 5′-Phosphate-Dependent Exonuclease (Lucigen). Finally, Cap-Clip (CellScript) was added to remove capped transcripts. Of the final uncapped fragmented mRNA preparation, 10% was reserved as input material, and the remaining 90% was subjected to immunoprecipitation with anti-m6A antibody (Abcam, ab151230). Immunoprecipitated RNA was eluted with RLT buffer (QIAGEN, 160051456), purified by ethanol precipitation, and prepared for library generation using a TruSeq mRNA library preparation kit (Illumina). Sequencing was performed at IGM Genomics Center, UCSD, using an Illumina NovaSeq 6000.
For m6Am-exo-qPCR, the same procedure as for m6Am-exo-seq was used through the anti-m6Am immunoprecipitation and RNA elution step. The eluted RNA and input samples were reverse transcribed and subjected to qPCR on a LightCycler 480 (Roche Diagnostics) using the primers listed in SI Appendix, Table S4.
Wang L., Wang S., Wu L., Li W., Bray W., Clark A.E., Gonzalez G.M., Wang Y., Carlin A.F, & Rana T.M. (2023). PCIF1-mediated deposition of 5′-cap N6,2′-O-dimethyladenosine in ACE2 and TMPRSS2 mRNA regulates susceptibility to SARS-CoV-2 infection. Proceedings of the National Academy of Sciences of the United States of America, 120(5), e2210361120.
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2

Quantitative Analysis of mRNA Cap Modifications

Cited 2 times
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mRNA was purified from control and PCIF1‐depleted cells using a Magnetic mRNA Isolation Kit (New England Biolabs, S1550S) according to the manufacturer's protocol. Aliquots of 0.5 μg mRNA were de‐capped with 1 U Cap‐Clip (CellScript) at 37°C for 1 h and digested with 0.5 U of nuclease P1 (Sigma) at 37°C for 2 h. Ammonium bicarbonate and alkaline phosphatase (Sigma) were added and the mixture was incubated at 37°C for 2 h. The samples were then filtered (0.22‐μm pore, Millipore) and analyzed by LC–MS/MS as previously described (Li et al, 2020 (link); Huff et al, 2021 (link)).
Wang L., Wu L., Zhu Z., Zhang Q., Li W., Gonzalez G.M., Wang Y, & Rana T.M. (2022). Role of PCIF1‐mediated 5′‐cap N6‐methyladeonsine mRNA methylation in colorectal cancer and anti‐PD‐1 immunotherapy. The EMBO Journal, 42(2), e111673.
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3

Transcription Start Site Sequencing

Cited 1 time
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We performed TSS-seq on 14-day-old seedlings grown on MS plates following a previously published protocol44 (link),65 (link). We applied heat stress by treating plants for 2 h at 37 °C. 5 µg of total RNA were treated with DNase and CIP (NEB) to remove DNA and all non-capped RNA. Then 5’ caps of capped RNA were removed with Cap-Clip (CellScript). We then ligated single-stranded rP5_RND adapters to 5’-ends with T4 RNA ligase 1 (NEB). Ligated RNAs were enriched and captured by oligo(dT) Dynabeads (Thermo Fisher Scientific). We fragmented Enriched samples for 5 min at 80 °C and generated the first-strand cDNA with SuperScript III (Invitrogen) and random primers. Second-strand cDNA was synthesized with Phusion High-Fidelity DNA Polymerase (NEB) and the BioNotI-P5-PET oligo and captured by Dynabeads for end repairing with End Repair Enzyme Mix (NEB) and ligation with barcoded Illumina compatible adapter using T4 DNA Ligase (NEB). We amplified TSS-seq sequencing libraries and size selected for single-end sequencing with NovaSeq 6000 platform (Illumina).
Zhong Z., Wang Y., Wang M., Yang F., Thomas Q.A., Xue Y., Zhang Y., Liu W., Jami-Alahmadi Y., Xu L., Feng S., Marquardt S., Wohlschlegel J.A., Ausin I, & Jacobsen S.E. (2022). Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis. Nature Communications, 13, 6970.
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4

HPLC-MS/MS Analysis of Decapped mRNA

Cited 2 times
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mRNA was purified from cells using a Magnetic mRNA Isolation Kit (New England Biolabs, S1550S) according to the manufacturer’s protocol. Aliquots (0.5 μg) of purified mRNA were de-capped by incubation with 1 U Cap-Clip (CellScript) at 37 °C for 60 min and then digested with 0.5 U nuclease P1 (Sigma) at 37 °C for 2 h. Ammonium bicarbonate and alkaline phosphatase (Sigma) were added, and the mixtures were incubated at 37 °C for an additional 2 h. All samples were then filtered (0.22-µm pore, Millipore) and analyzed by HPLC-MS/MS as previously described (21 (link), 26 (link)).
Wang L., Wang S., Wu L., Li W., Bray W., Clark A.E., Gonzalez G.M., Wang Y., Carlin A.F, & Rana T.M. (2023). PCIF1-mediated deposition of 5′-cap N6,2′-O-dimethyladenosine in ACE2 and TMPRSS2 mRNA regulates susceptibility to SARS-CoV-2 infection. Proceedings of the National Academy of Sciences of the United States of America, 120(5), e2210361120.
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5

m6Am-exo-Seq and m6Am-exo-qPCR Protocol

Cited 1 time
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m6Am‐exo‐Seq was performed according to previously described protocols (Sendinc et al, 2019 (link)). Briefly, mRNA was purified from control and PCIF1‐depeleted HCT116 cells using a Magnetic mRNA Isolation Kit (New England Biolabs, S1550S). mRNA was then fragmented with a Fragmentation Reagents Kit (Invitrogen, AM8740), phosphorylated with T4 PNK (NEB, M0201S), and treated with Terminator 5′‐Phosphate‐Dependent Exonuclease (Lucigen) to remove phosphorylated transcripts. Lastly, Cap‐Clip (CellScript) was used to remove capped transcripts. A sample equivalent to 10% of the 5′‐uncapped mRNA fragments was reserved as input, and the remainder was immunoprecipitated with anti‐m6A antibody (Abcam, ab151230). Library generation and sequencing were performed at IGM Genomics core, UCSD on an Illumina NovaSeq 6000.
For m6Am‐exo‐qPCR, mRNA was extracted from freshly excised control and Pcif1‐depeleted tumors and processed using the procedures described above. Reserved input and anti‐m6Am immunoprecipitated samples were reverse‐transcribed and analyzed by qPCR as described above.
Wang L., Wu L., Zhu Z., Zhang Q., Li W., Gonzalez G.M., Wang Y, & Rana T.M. (2022). Role of PCIF1‐mediated 5′‐cap N6‐methyladeonsine mRNA methylation in colorectal cancer and anti‐PD‐1 immunotherapy. The EMBO Journal, 42(2), e111673.
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6

Isolation and Analysis of Small RNAs from HepG2 Cells

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Human liver hepatocellular carcinoma cells (HepG2) were grown, and RNA was isolated with TRI-Reagent (Sigma). After DNase treatment and RNA integrity check by denaturating gel electrophoresis, sRNA fractions were enriched by urea tris-boric-acid EDTA polyacrylamide gel electrophoreses between 19 and 31 nucleotides and fluorometrically quantified. For pyrophosphatase treatment, 10 g of RNA treated with CapClip™ (Cellscript) and RNase inhibitor was purified with acid phenol. For controls, CapClip reactions were split and the spike in oligo was added to one sample. The 5Ј-triphosphorylated control oligo was transcribed in vitro from 2 annealed DNAs. Parallel samples without spike in were used for sequencing. Success of the treatment was monitored by subsequent treatment with Ter-minator™ 5Ј-monophosphate-specific RNase (Epicentre). Samples before and after Terminator treatment were visualized by denaturing polyacrylamide gel electrophoreses (see Fig. 1 in the online Data Supplement). Quantitative reverse transcription PCR (qRT-PCR) was carried out after reverse transcription with specific hairpin primers as described in (19 ) ; relative enrichment was calculated with a spike in RNA oligo and internal miRNAs by application of the E ⌬⌬Cp method (Eamplification efficiency E ϭ 10 Ϫ1/slope ). Details can be found in Fig. 2 in the online Data Supplement.
Meistertzheim M., Fehlmann T., Drews F., Pirritano M., Gasparoni G., Keller A, & Simon M. (2019). Comparative Analysis of Biochemical Biases by Ligation- and Template-Switch-Based Small RNA Library Preparation Protocols. Clinical chemistry, 65(12).
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