title: Small RNA library construction from minute biological samples.
abstract: Increasingly, the discovery and characterization of small regulatory RNAs from a variety of organisms have all required deep-sequencing methodologies. However, the crux to successful deep-sequencing analysis depends upon optimal construction of a cDNA library compatible for the high-throughput sequencing platform. Challenges to small RNA library constructions arise when dealing with minute tissue samples because certain structural RNA fragments can dominate and mask the desired characterization of regulatory small RNAs like microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs). Here, we describe methods that improve the chances of constructing a successful library from small RNAs isolated from minute tissues such as enriched follicle cells from the Drosophila ovarium. Because the ribosomal RNA (rRNA) fragments are frequently the major contaminants in small RNA preparations from minute amounts of tissue, we demonstrate the utility of antisense oligonucleotide depletion and an acryloylaminophenylboronic acid (APB) polyacrylamide gel system for separating the abundant 2S rRNA in Drosophila from endo-siRNAs and piRNAs. Finally, our methodology generates libraries amenable to multiplex sequencing on the Illumina Hi-Seq platform.
authors.affiliation: Department of Biology, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, MA, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Matts
authors.forename: Yuliya
authors.initials: Y
authors.lastname: Sytnikova
authors.forename: Gung-Wei
authors.initials: GW
authors.lastname: Chirn
authors.forename: Gabor L
authors.initials: GL
authors.lastname: Igloi
authors.forename: Nelson C
authors.initials: NC
authors.lastname: Lau
mesh_terms: D000818:Animals; D001897:Boronic Acids / Q000737:chemistry; D004331:Drosophila melanogaster / Q000166:cytology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D020320:Oligoribonucleotides, Antisense / Q000235:genetics; D009928:Organ Specificity; D006080:Ovarian Follicle / Q000166:cytology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA; D013048:Specimen Handling
chemical_list: D001897:Boronic Acids; D020320:Oligoribonucleotides, Antisense; D034741:RNA, Small Interfering