By Andreas Werner
Animal Endo-SiRNAs: equipment and Protocols offers quite a few methods to enquire endo-siRNAs. those contain protocols appropriate to review brief RNAs expressed at a low point and version platforms which are rather compatible to enquire particular facets of endo-siRNAs, their synthesis, their genomics or regulatory function. Written within the hugely profitable Methods in Molecular Biology series layout, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, comfortably reproducible laboratory protocols and tips about troubleshooting and heading off identified pitfalls.
Authoritative and useful, Animal Endo-SiRNAs: tools and Protocols includes useful assistance which are absent in normal lab manuals.
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If you have enough material, you can consider splitting the sample and use one part for cloning and the other for β-elimination. 3. Use 1 μL of the final product for quality control on a 20 % denaturing acrylamide/urea gel (a mini-gel setup as used for western blots will do just fine). After staining with Sybr gold, the RNA bands can be visualized and documented. In Drosophila RNA, shortening of the 30 nt long 2S rRNA can be easily documented if an untreated and a β-eliminated sample are loaded side by side.
9. , Alexa Fluor 594 anti-rabbit IgG, 1:500) for 1 h at room temperature. 10. 1 % Tween-20 followed by a rinse with PBS. 11. Mount in mounting medium with DAPI (4′,6-diamidino-2phenylindole). 4 Notes 1. Instead of Protein G you can use other surface functionalities to covalently couple the primary antibody to the Dynabeads. However, in our hands other strategies to couple the antibody to the Dynabeads result in lower yields of immunoprecipitated CBs. 2. Alternatively, PBS can be used for antibody binding and washing steps.
Isopropanol. 10. Dimethyl sulfoxide (DMSO). 11. Truncated T4 RNA ligase 2 with the supplied buffer (NEB). 12. (Normal) T4 RNA ligase 1 with the supplied buffer (NEB). 13. Reverse transcriptase including 5× first strand buffer and 100 mM DTT. 14. RNase inhibitor (for example RNasin from Promega). 15. Hot Start Phusion polymerase including 5× reaction buffer. 16. dNTP mix (10 mM each). 17. Gel Extraction Kit (Qiagen). 18. Required oligonucleotides: First ligation at the 3′-end: Linker 1 AMP-5′p = 5′pCTGTAGGCACCATCAATdideoxyC-3′ Pre-adenylated linker, commercially available from IDT Product name: miRNA cloning linker 1 Prepare aliquots of 50 μM stock solution Second ligation at the 5′-end: Illumina 5′-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrC linker rUrCrUrUrCrCrGrArUrCrU-3′ Our oligonucleotide supplier is MWG/Eurofins Quality: HPLC purified, prepare aliquots of 50 μM stock solution Deep Sequencing of Endo-siRNAs 39 Reverse transcription: RT primer 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC index GATTGATGGTGCCTACAG-3′ MWG—Custom oligo—HPSF purified, 5 μM stock (underlined: Illumina index primer binding site) PCR amplification: 5′-Illumina paired 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCT ACACGACG-3′ (underlined sequence: necessary for compatibility with paired-end flow cells on the Illumina systems) MWG—Custom oligo—HPSF purified, aliquots of 10 μM stock 3’-PCR index1 5'CAAGCAGAAGACGGCATACGAGATatcacgGTGACTGGAGTTCA GACGTG -3' 3’-PCR index2 5'CAAGCAGAAGACGGCATACGAGATcgatgtGTGACTGGAG TTCAGACGTG -3' 3’-PCR index3 5'- 3’-PCR index4 TTCAGACGTG -3' 5'- 3’-PCR index5 TTCAGACGTG -3' 5'- 3’-PCR index6 TTCAGACGTG -3' 5'- 3’-PCR index7 TTCAGACGTG -3' 5’- 3’-PCR index8 TTCAGACGTG -3’ 5’- CAAGCAGAAGACGGCATACGAGATttaggcGTGACTGGAG CAAGCAGAAGACGGCATACGAGATtgaccaGTGACTGGAG CAAGCAGAAGACGGCATACGAGATcagaatGTGACTGGAG CAAGCAGAAGACGGCATACGAGATgctgtaGTGACTGGAG CAAGCAGAAGACGGCATACGAGATtcgcacGTGACTGGAG CAAGCAGAAGACGGCATACGAGATcagtggGTGACTGGAG TTCAGACGTG -3’ 3’-PCR index9 5’CAAGCAGAAGACGGCATACGAGATggtatcGTGACTGGAG TTCAGACGTG -3’ All index primers: MWG/Eurofins, HPSF purified, aliquots of 10 μM stock (underlined sequence: necessary for compatibility with paired-end flow cells on the Illumina systems) 40 4 Katharina Elmer et al.
Animal Endo-SiRNAs: Methods and Protocols by Andreas Werner