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Genomewide characterization of non-polyadenylated RNAs

Li Yang13, Michael O Duff1, Brenton R Graveley1, Gordon G Carmichael1 and Ling-Ling Chen12*

Author Affiliations

1 Department of Genetics and Developmental Biology, University of Connecticut Stem Cell Institute, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030-6403, USA

2 State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China

3 Current address: Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China

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Genome Biology 2011, 12:R16  doi:10.1186/gb-2011-12-2-r16

Published: 16 February 2011

Additional files

Additional file 1:

Correlation among poly(A)+ and poly(A)- samples in H9 and HeLa cells. The scale for all plots is log10. 1-3, the sequencing triplicates of sample poly(A)- transcripts in H9 cells; 4-6, the sequencing triplicates of sample poly(A)+ transcripts in H9 cells; 7-9, the sequencing triplicates of sample poly(A)- transcripts in HeLa cells; 10-12, the sequencing triplicates of sample poly(A)+ transcripts in HeLa cells. Note the linear correlation among triplicates of individual samples is greater than 98%.

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Additional file 2:

Read counts and coverage per concatenated sample.

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Additional file 3:

Full list of genes and deep sequencing results from HeLa cells. Reads from poly(A)- (#1) and poly(A)+ (#2) samples are normalized for BPKM. Genes are sorted by their Wald scores (WaldStat).

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Additional file 4:

Full list of genes and deep sequencing results from H9 cells. Reads from poly(A)- (#1) and poly(A)+ (#2) samples are normalized for BPKM. Genes are sorted by their Wald scores (WaldStat).

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Additional file 5:

List of poly(A)- genes from HeLa cells. Criteria for inclusion in this list are described in the text. Normalized reads from poly(A)- and poly(A)+ samples. Genes are sorted by their Wald scores (WaldStat).

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Additional file 6:

List of poly(A)- genes from H9 cells. Criteria for inclusion in this list are described in the text. Reads from poly(A)- and poly(A)+ samples are normalized. Genes are sorted by the Wald score (WaldStat).

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Additional file 7:

Malat1 and neat1 are examples of poly(A)- and bimorphic long non-coding RNAs. (a) Malat1 exists in both poly(A)+ and poly(A)- isoforms and the poly(A)- isoform is more abundant than the poly(A)+ isoform. Fewer reads from the 5' end in the poly(A)- fraction are aligned to the genome in both cell lines. Malat1 is also more abundantly expressed in HeLa cells than in H9 cells. (b) Semi-quantitative RT-PCR with two sets of primers confirmed that malat1 is more abundant in poly(A)- samples. (c) Deep sequencing reveals that both isoforms of neat1 are undetectable in H9 cells. In HeLa cells, while the shorter isoform of neat1 is entirely poly(A)+ (pink color), the longer isoform is more enriched in the poly(A)- fraction (red), also seen in (d) for its relative abundance with the same y-axis in HeLa cells.

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Additional file 8:

List of bimorphic genes from HeLa cells. Criteria for inclusion in this list are described in the text. Reads from poly(A)- and poly(A)+ samples are normalized. Genes are sorted by their Wald scores (WaldStat).

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Additional file 9:

List of bimorphic genes from H9 cells. Criteria for inclusion in this list are described in the text. Reads from poly(A)- and poly(A)+ samples are normalized. Genes are sorted by their Wald scores (WaldStat).

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Additional file 10:

List of bimorphic genes that overlap in both H9 and HeLa cells.

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Additional file 11:

The 3' end of the shorter isoform of h2afx contains the canonical consensus sequence within the 3' UTR of non-polyadenylated histone genes. (a) MEME analysis (Multiple Em for Motif Elicitation) [22,23] revealed the consensus sequence within the 3' UTR regions of histone genes for poly(A)- RNAs. (b) MFold analysis (version 3.5, M Zuker, Rensselaer Polytechnic Institute) predicted the stem-loop structure within the 3' UTR of histone genes for poly(A)- RNAs.

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Additional file 12:

Visualization of transcripts exhibiting 3' decay in poly(A)- samples. (a) Examples of 3' decay in the bimorphic group. pdk 4 (pyruvate dehydrogenase kinase, isozyme 4) is expressed in HeLa cells only and shows similar normalized read densities in poly(A)+ and poly(A)- samples; however, it exhibits a gradual decay pattern (blue dashed lines) from 3' to 5' in the poly(A)- sample in HeLa cells. (b-d) Examples of 3' decay in the poly(A)+ group. Note that sfrs18 (splicing factor, arginine/serine-rich 18), sf3b2 (splicing factor 3b, subunit 2) and eif3a (eukaryotic translation initiation factor 3) all exhibit a gradual decay pattern (blue dashed lines) from the 3' to 5' ends in poly(A)- samples in both H9 and HeLa cells, although both are more abundant in poly(A)+ samples. See text for details.

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Additional file 13:

Excised introns. (a) The 12th intron of smpd4 (sphingomyelin phosphodiesterase 4) mRNA is an excised intron (blue dashed box) and accumulates in the poly(A)- sample in H9 cells. (b) The second intron of atad3b (ATPase family, AAA domain containing 3B) mRNA accumulates in the poly(A)- samples in both H9 and HeLa cells (blue dashed box).

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Additional file 14:

Poly(A)- exons. (a) The second and third exons of camsap1 (calmodulin regulated spectrin-associated protein 1) mRNA accumulate in poly(A)- samples from both H9 and HeLa cells (blue dashed box). (b) Examples of poly(A)- exons in mRNAs, and the positions of the exons in mRNAs are indicated.

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Additional file 15:

List of poly(A)- genes that overlap in both H9 and HeLa cells.

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Additional file 16:

Histone mRNAs preferentially expressed in H9 cells (hist1h1d, hist1h3i, and hist1h3j; black) or HeLa cells (hist1h3c; red). Only unique alignments were allowed. See Figure 6c for the relative transcription of these genes upon differentiation or reprogramming.

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Additional file 17:

A schematic view of histone gene cluster 1 on chromosome 6 comparing the expression of histone genes in H9 and HeLa cells. While most histone genes (hist1h2bl, hist1h2ai, hist1h3h, hist1h2ai, hist1h2bm, hist1h2ak, hist1h2bn, hist1h2am and hist1h2bo) are expressed in both cell lines, some histone genes (hist1h2al, hist1h1b, hist1h3i, hist1h4l and hist1h3j) are expressed at significantly higher levels in H9 cells.

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Additional file 18:

Gene-specific primers used for RT-PCR and qPCR validation.

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