Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80–118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3′ end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets.
References
[1]
Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, et al. (2005) The transcriptional landscape of the mammalian genome. Science 309: 1559–1563.
[2]
Kapranov P, Cawley SE, Drenkow J, Bekiranov S, Strausberg RL, et al. (2002) Large-scale transcriptional activity in chromosomes 21 and 22. Science 296: 916–919.
[3]
Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, et al. (2005) Antisense transcription in the mammalian transcriptome. Science 309: 1564–1566.
[4]
Mattick JS, Taft RJ, Faulkner GJ (2010) A global view of genomic information–moving beyond the gene and the master regulator. Trends in genetics : TIG 26: 21–28.
Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10: 126–139.
[7]
Matera AG, Terns RM, Terns MP (2007) Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nat Rev Mol Cell Biol 8: 209–220.
[8]
Mancini-Dinardo D, Steele SJ, Levorse JM, Ingram RS, Tilghman SM (2006) Elongation of the Kcnq1ot1 transcript is required for genomic imprinting of neighboring genes. Genes Dev 20: 1268–1282.
[9]
Sleutels F, Zwart R, Barlow DP (2002) The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415: 810–813.
[10]
Williamson CM, Ball ST, Dawson C, Mehta S, Beechey CV, et al. (2011) Uncoupling antisense-mediated silencing and DNA methylation in the imprinted Gnas cluster. PLoS genetics 7: e1001347.
[11]
Wilhelm BT, Landry JR (2009) RNA-Seq-quantitative measurement of expression through massively parallel RNA-sequencing. Methods 48: 249–257.
[12]
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10: 57–63.
[13]
Metzker ML (2010) Sequencing technologies - the next generation. Nat Rev Genet 11: 31–46.
[14]
Pepke S, Wold B, Mortazavi A (2009) Computation for ChIP-seq and RNA-seq studies. Nat Methods 6: S22–32.
[15]
Costa V, Angelini C, De Feis I, Ciccodicola A (2010) Uncovering the complexity of transcriptomes with RNA-Seq. J Biomed Biotechnol 2010: 853916.
[16]
Roberts A, Trapnell C, Donaghey J, Rinn JL, Pachter L (2011) Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol 12: R22.
[17]
Oshlack A, Wakefield MJ (2009) Transcript length bias in RNA-seq data confounds systems biology. Biol Direct 4: 14.
[18]
Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, et al. (2006) The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol 24: 1151–1161.
[19]
Cloonan N, Forrest AR, Kolle G, Gardiner BB, Faulkner GJ, et al. (2008) Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods 5: 613–619.
[20]
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5: 621–628.
[21]
Ramskold D, Wang ET, Burge CB, Sandberg R (2009) An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data. PLoS Comput Biol 5: e1000598.
[22]
Cui P, Lin Q, Ding F, Xin C, Gong W, et al. (2010) A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing. Genomics 96: 259–265.
[23]
Chen AC, Gudas LJ (1996) An analysis of retinoic acid-induced gene expression and metabolism in AB1 embryonic stem cells. J Biol Chem 271: 14971–14980.
[24]
t Hoen PA, Ariyurek Y, Thygesen HH, Vreugdenhil E, Vossen RH, et al. (2008) Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms. Nucleic Acids Res 36: e141.
[25]
Armour CD, Castle JC, Chen R, Babak T, Loerch P, et al. (2009) Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nature methods 6: 647–649.
[26]
Castle JC, Armour CD, Lower M, Haynor D, Biery M, et al. (2010) Digital genome-wide ncRNA expression, including SnoRNAs, across 11 human tissues using polyA-neutral amplification. PloS one 5: e11779.
[27]
Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, et al. (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320: 1344–1349.
[28]
Pesole G, Mignone F, Gissi C, Grillo G, Licciulli F, et al. (2001) Structural and functional features of eukaryotic mRNA untranslated regions. Gene 276: 73–81.
[29]
Schwartz S, Oren R, Ast G (2011) Detection and removal of biases in the analysis of next-generation sequencing reads. PLoS One 6: e16685.
[30]
Xu G, Fewell C, Taylor C, Deng N, Hedges D, et al. (2010) Transcriptome and targetome analysis in MIR155 expressing cells using RNA-seq. RNA 16: 1610–1622.
[31]
Houbaviy HB, Murray MF, Sharp PA (2003) Embryonic stem cell-specific MicroRNAs. Dev Cell 5: 351–358.
[32]
Karali M, Peluso I, Marigo V, Banfi S (2007) Identification and characterization of microRNAs expressed in the mouse eye. Invest Ophthalmol Vis Sci 48: 509–515.
[33]
Latos PA, Stricker SH, Steenpass L, Pauler FM, Huang R, et al. (2009) An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias. Development 136: 437–448.
[34]
Mercer TR, Dinger ME, Sunkin SM, Mehler MF, Mattick JS (2008) Specific expression of long noncoding RNAs in the mouse brain. Proc Natl Acad Sci U S A 105: 716–721.
[35]
Koerner MV, Pauler FM, Huang R, Barlow DP (2009) The function of non-coding RNAs in genomic imprinting. Development 136: 1771–1783.
[36]
Furuno M, Pang KC, Ninomiya N, Fukuda S, Frith MC, et al. (2006) Clusters of internally primed transcripts reveal novel long noncoding RNAs. PLoS Genet 2: e37.
[37]
Pandey RR, Mondal T, Mohammad F, Enroth S, Redrup L, et al. (2008) Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell 32: 232–246.
[38]
Pauler FM, Stricker SH, Warczok KE, Barlow DP (2005) Long-range DNase I hypersensitivity mapping reveals the imprinted Igf2r and Air promoters share cis-regulatory elements. Genome Res 15: 1379–1387.
[39]
Marks H, Chow JC, Denissov S, Francoijs KJ, Brockdorff N, et al. (2009) High-resolution analysis of epigenetic changes associated with X inactivation. Genome Res 19: 1361–1373.
[40]
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25.
[41]
Lyle R, Watanabe D, te Vruchte D, Lerchner W, Smrzka OW, et al. (2000) The imprinted antisense RNA at the Igf2r locus overlaps but does not imprint Mas1. Nat Genet 25: 19–21.
