Abstract
Small RNA sequencing by Illumina’s Next Generation technology has revolutionized the transcriptome analysis by facilitating massive parallel sequencing of RNA molecules at low cost. Illumina’s Next Generation RNA sequencing is ideal for profiling small RNA (microRNAs, snoRNAs, and piRNAs) libraries in the identification of novel biomarkers for better clinical diagnosis. This method offers significant advantages when compared to microarray analysis with the ability to identify novel transcripts, higher sensitivity, specificity, and detection of rare and low-abundance transcripts. Small RNAs, including microRNAs and snoRNAs, belong to the class of small non-coding RNAs with 50–200 nucleotides in length and are involved in post-transcriptional regulation of gene expression. Executing Illumina’s Next Generation Sequencing technology, we have recently deciphered microRNAs and snoRNAs expressed in cerebral cavernous malformations (CCMs). Small RNA library preparation is a prerequisite step prior to RNA sequencing for the identification of microRNAs and snoRNAs. Here, we describe stepwise small RNA library preparation starting from total RNA isolated from CCMs patient until library validation using the Illumina® TruSeq® Small RNA Sample preparation kit. We believe this method will shed light into the functional identification of other novel small non-coding RNAs in CCMs that awaits discovery.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Fisher OS, Zhang R, Li X, Murphy JW, Demeler B, Boggon TJ (2013) Structural studies of cerebral cavernous malformations 2 (CCM2) reveal a folded helical domain at its C-terminus. FEBS Lett 587(3):272–277
Kar S, Bali KK, Baisantry A, Geffers R, Samii A, Bertalanffy H (2017) Genome-wide sequencing reveals microRNAs downregulated in cerebral cavernous malformations. J Mol Neurosci 61(2):178–188
Schneider H, Errede M, Ulrich NH, Virgintino D, Frei K, Bertalanffy H (2011) Impairment of tight junctions and glucose transport in endothelial cells of human cerebral cavernous malformations. J Neuropathol Exp Neurol 70(6):417–429
Kar S, Bali KK, Baisantry A, Geffers R, Hartmann C, Samii A, Bertalanffy H (2018) Genome-wide sequencing reveals small nucleolar RNAs downregulated in cerebral cavernous malformations. Cell Mol Neurobiol 38(7):1369–1382
Koskimaki J, Girard R, Li Y, Saadat L, Zeineddine HA, Lightle R, Moore T, Lyne S, Avner K, Shenkar R, Cao Y, Shi C, Polster SP, Zhang D, Carrion-Penagos J, Romanos S, Fonseca G, Lopez-Ramirez MA, Chapman EM, Popiel E, Tang AT, Akers A, Faber P, Andrade J, Ginsberg M, Derry WB, Kahn ML, Marchuk DA, Awad IA (2019) Comprehensive transcriptome analysis of cerebral cavernous malformation across multiple species and genotypes. JCI Insight 4(3)
Bai Y, Zhang Z, Jin L, Kang H, Zhu Y, Zhang L, Li X, Ma F, Zhao L, Shi B, Li J, McManus DP, Zhang W, Wang S (2014) Genome-wide sequencing of small RNAs reveals a tissue-specific loss of conserved microRNA families in Echinococcus granulosus. BMC Genomics 15:736
Shore S, Henderson JM, Lebedev A, Salcedo MP, Zon G, McCaffrey AP, Paul N, Hogrefe RI (2016) Small RNA library preparation method for next-generation sequencing using chemical modifications to prevent adapter dimer formation. PLoS One 11(11):e0167009
Moore LM, Kivinen V, Liu Y, Annala M, Cogdell D, Liu X, Liu CG, Sawaya R, Yli-Harja O, Shmulevich I, Fuller GN, Zhang W, Nykter M (2013) Transcriptome and small RNA deep sequencing reveals deregulation of miRNA biogenesis in human glioma. J Pathol 229(3):449–459
Li P, Shen M, Gao F, Wu J, Zhang J, Teng F, Zhang C (2017) An Antagomir to microRNA-106b-5p Ameliorates cerebral ischemia and reperfusion injury in rats via inhibiting apoptosis and oxidative stress. Mol Neurobiol 54(4):2901–2921
Ferreira R, Santos T, Amar A, Tahara SM, Chen TC, Giannotta SL, Hofman FM (2014) MicroRNA-18a improves human cerebral arteriovenous malformation endothelial cell function. Stroke 45(1):293–297
Orso F, Balzac F, Marino M, Lembo A, Retta SF, Taverna D (2013) miR-21 coordinates tumor growth and modulates KRIT1 levels. Biochem Biophys Res Commun 438(1):90–96
Yeri A, Courtright A, Danielson K, Hutchins E, Alsop E, Carlson E, Hsieh M, Ziegler O, Das A, Shah RV, Rozowsky J, Das S, Van Keuren-Jensen K (2018) Evaluation of commercially available small RNASeq library preparation kits using low input RNA. BMC Genomics 19(1):331
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kar, S., Geffers, R., Samii, A., Bertalanffy, H. (2020). Library Preparation for Small RNA Transcriptome Sequencing in Patients Affected by Cerebral Cavernous Malformations. In: Trabalzini, L., Finetti, F., Retta, S. (eds) Cerebral Cavernous Malformations (CCM) . Methods in Molecular Biology, vol 2152. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0640-7_35
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0640-7_35
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0639-1
Online ISBN: 978-1-0716-0640-7
eBook Packages: Springer Protocols