miRBase name tgu-let-7a tgu-let-7f tgu-miR-124 tgu-miR-9 tgu-miR-129-5p tgu-miR-129-3p tgu-miR-29a tgu-miR-92 tgu-miR-25 RNU6B tgu-miR-2954-5p tgu-miR-2954-3p tgu-miR-2954R-5p tgu-miR-2954R-3p let-7a let-7f miR-124 miR-9 miR-129-5p miR-129-3p miR-29c miR-92a miR-25 RNU6B novel51F-5p novel51F-3p novel51R-5p novel51R-3p 5’-UGAGGUAGUAGGUUGUAUAGUU-3’ 5’-UGAGGUAGUAGAUUGUAUAGUU-3’ 5’-UAAGGCACGCGGUGAAUGCC-3’ 5’-UCUUUGGUUAUCUAGCUGUAUGA-3’ 5’-CUUUUUGCGGUCUGGGCUUGC-3’ 5’-AAGCCCUUACCCCAAAAAGCAU-3’ 5’-UAGCACCAUUUGAAAUCGGU-3’ 5’-UAUUGCACUUGUCCCGGCCUGU-3’ 5’-CAUUGCACUUGUCUCGGUCUGA-3’ 5’-CGCAAGGAUGACACGCAAAUUCGUGAAGCGUUCCAUAUUUUU-3’ 5’-GCUGAGAGGGCUUGGGGAGAGGA-3’ 5’-CAUCCCCAUUCCACUCCUAGCA-3’ (Northern validated) 5’-UGCUAGGAGUGGAAUGGGGAUG-3’ 5’-UCCUCUCCCCAAGCCCUCUCAGC-3’ Gunaratne et al. BMC Genomics 2011, 12:277 http://www.biomedcentral.com/1471-2164/12/277 Page 11 of 14 Table 4 Probes used for Taqman analysis of specific miRNA sequences Company name Sequence detected min at 200V using 1X TBE buffer at room temperature. The membrane was cross-linked at 1200 kJ for 45 sec- onds. RNA probes were synthesized for tgu-miR-2954-3p probe 5’ - UGCUAGGAGUGGAAUGGGGAU G - 3’ by Integrated DNA Technologies. Radio labeling was carried out in a reaction of 12.0ul dH2O + 2.0ul PNK buffer + 1.0ul (100ng/ul) probe + 1.0ul PNK polymerase (Pro- mega) + 4.0ul P32-gamma-ATP (10mCi/ml) (PerkinEl- mer). The reaction was incubated at 37°C for 1 hour and inactivated at 65°C for 10 min. The probe was purified using Nick columns from GE following manufacturer’s instructions. The membranes were pre-hybridized for 30 min with 20 ml of pre-hybridization buffer (5X SSC + 20 mM NaPO4 + 7X SDS + 2X Denhardt (pre warmed) at 60° C) in a rotating hybridization oven. Hybridization was carried out at 50°C in a rotating incubator for 24h. The membranes were washed for 10 min at 50°C with 20-30mL of wash buffer (2X SSC + 0.5% SDS). When background was ~0.5 cpm, the membranes were wrapped in saran wrap and exposed at -80°C for ~72h. Additional material Additional file 1: Supplemental tables.xls. This one file contains all four Supplemental Tables, each as a separate worksheet. Table S1 ("1 overview”) is a summary of Illumina sequence read alignments for six pools of RNA from zebra finch auditory forebrain responding to song versus silence, and shows the distribution of sequence reads in relation to multiple genomes and multiple annotations in the current genomic databases. Table S2 ("2 novel hairpins”) gives detailed alignments of putative pre-miRNAs and read sequences. Table S3 ("3 novel genes”) shows annotations of all novel miRNA loci mapped in genome assemblies of zebra finch, chicken or human. Table S4 ("4 all read counts”) gives read counts and current annotation in miRBase of all conserved and novel miRNAs, with statistics. Additional file 2: Supplemental figures.doc. This one file contains all three supplemental figures. Figure S1 is a Venn diagram of numbers of miRNAs with significant differential expression in response to novel song in three Illumina experiments. Figure S2 shows a comparative mapping in other avian transcriptomes of tgu-mir-2954. Figure S3 demonstrates the song-specificity of the miRNA response, using TaqMan to compare the levels of specific miRNAs in animals from groups that heard song, matching song-enveloped noise, or silence. Acknowledgements We thank Sarah London for useful discussions and contributions to the text. Supported by NIH RO1 NS045264 and RO1 NS051820 (to D.F.C). Note Added in Proof: The novel miRNA referred to here as “miR-2954-3p” is now identified in miRBase as “miR-2954”. The novel miRNA referred to here as “miR-2954-5p” is now identified in miRBase as “miR-2954*”. Author details 1Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204, USA. 2Departments of Pathology, Baylor College of Medicine, Houston, Texas 77030, USA. 3Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas 77030, USA. 4Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, IL 61801, USA. 5W.M. Keck Center for Comparative and Functional Genomics, Roy J. Carver Biotechnology Center, University of Illinois, Urbana-Champaign, IL 61801, USA. 6Dan Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA. 7ARK-Genomics, The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, EH25 9RG, UK. 8Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK. 9Institute for Genomic Biology, University of Illinois, Urbana-Champaign, IL 61801, USA. 10Beckman Institute, University of Illinois, Urbana-Champaign, IL 61801, USA. 11Bioinformatics Research Laboratory (BRL), Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. Authors’ contributions PHG coordinated the work of Illumina RNA-seq and prepared the manuscript. YL conducted the song exposure experiments and subsequent dissections and RNA extractions, performed TaqMan qPCR, analyzed differentially expressed miRNAs and participated in drafting the manuscript. ALB performed Illumina RNA-seq and Northern blot. JD helped analyze expression data of Illumina RNA-seq and TaqMan qPCR. CC, JBT, CJC, JHK, and AM participated in mapping and analyzing Illumina RNA-seq data. MW and SGJ helped with miRNA sequence annotation. DFC designed and coordinated the study and drafted the manuscript. All authors read and approved the manuscript. Received: 28 July 2010 Accepted: 31 May 2011 Published: 31 May 2011