By Andreas Werner

ISBN-10: 1493909304

ISBN-13: 9781493909308

ISBN-10: 1493909312

ISBN-13: 9781493909315

Animal Endo-SiRNAs: equipment and Protocols offers a number of ways to enquire endo-siRNAs. those comprise protocols acceptable to review brief RNAs expressed at a low point and version structures which are quite compatible to enquire particular features of endo-siRNAs, their synthesis, their genomics or regulatory function. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible laboratory protocols and pointers on troubleshooting and fending off recognized pitfalls.

Authoritative and useful, Animal Endo-SiRNAs: equipment and Protocols comprises useful tips which are absent in average lab manuals.

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Any indexes that cannot be interpreted and the corresponding sequence reads are collected in an additional file pair for unassigned reads. If any mistakes occurred during sample sheet editing, this file pair can subsequently be reinterpreted and de-multiplexed offline. We are happy to provide short PERL scripts that can be used for offline de-multiplexing, trimming of the 3′-linker sequence, and size-selection of the obtained small RNA sequences. It is usually advisable to exclude reads shorter than 16 nt from analysis as these may be difficult to be mapped unambiguously.

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.

Under special circumstances it may be possible to detect some of these siRNAs by low-sensitivity techniques like northern blotting; however, a representative view can only be obtained through deep sequencing. This technique was also at the origin of endo-siRNA discovery and mapping of the reads pointed to a biological role of these endosiRNAs: The repression of transcripts from transposable element in somatic and germ line cells. Comparable approaches have since identified other roles, such as chromatin modification [5–8], alternative splicing [9, 10], and recently DNA double-strand break repair [11–13].

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Animal Endo-SiRNAs: Methods and Protocols by Andreas Werner

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