BACKGROUND
the transcriptome is the complete catalogue of transcripts in the human cell.
at any given time, a wide range of different rna molecules are present at different levels of maturation and processing.
the rates and the dynamics of rna transcription and processing are unique for each cell.
previous studies have highlighted the importance of creating a complete map of transcripts, and the importance of understanding how different physiological conditions, developmental stages and disease can affect expression and regulation  <cit> .
in the recent years, rna-sequencing  has emerged as a standard procedure to study transcriptomes and measure levels of gene expression  <cit> .
studies using this method have significantly expanded our understanding of transcriptome complexity and provided new insights into the mechanisms of gene expression and transcriptional regulation in development and disease  <cit> .
however, many challenges still remain, primarily linked to data analysis and sample preparation  <cit> .
the starting material for rna-seq is typically total rna or polyadenylated  rna, which both represent heterogeneous pools of rna molecules at different stages of maturation and processing  <cit> .
several limitations arise from analyzing these populations of rnas from whole cells.
a commonly neglected problem is that total rna, but also polya+ rna to a lesser extent, contains substantial amounts of intronic rna originating from immature transcripts  <cit> .
this intronic background coverage is accentuated in long genes expressed at high levels, and is most noticeable in brain tissue where long neuronal genes tend to be highly expressed.
the presence of intronic rnas may influence the sensitivity to detect transcripts, identify splice junctions and measure gene expression levels, as a large proportion of sequence reads is mapped to introns  <cit> .
also, oligo-dt purification steps are likely to introduce certain biases, such as unspecific retrieval of rna containing poly-a stretches within the transcribed sequence, 5′ to 3′ biases, or truncated transcripts resulting either from alternative polyadenylation signals within introns or rna degradation products  <cit> .
although total rna-seq has been shown to provide insight into ongoing transcription and co-transcriptional splicing in the nucleus  <cit> , the simultaneous presence of mature rnas from the cytoplasm confounds the analysis of nuclear rna maturation steps.
recently, two technologies, the genome-wide nuclear run-on sequencing  and native elongating transcript sequencing , have been described to study nascent transcripts.
gro-seq yields an overview of transcription dynamics and directionality by labeling transcriptionally engaged nascent transcripts genome-wide, followed by high-throughput sequencing.
net-seq uses the stability of the ternary complex of dna, rna polymerase  and nascent rna to capture and sequence nascent transcripts in living cells using endogenously expressed rnapii with a 3 × −flag epitope.
these methods have successfully provided snapshots of ongoing transcription in cell lines  <cit> .
however, these methods do not provide any insight into posttranscriptional events, and are based upon manipulation of the normal physiological conditions of the cells and require extensive optimization and standardization.
recent improvements in rna extraction protocols now make it possible to study specific pools of rna molecules, either by fractionation of subcellular compartments or by molecular capture of specific rna-associated targets.
several kits for rna extraction from different cellular fractions are commercially available .
however, these kits are associated with significant amounts of cross contamination between the fractions.
to overcome the effects of cross contamination, recent studies used the selection of polya+ from the cytoplasmic fraction and chromatin-associated transcripts from the nuclear fraction to obtain more homogenous pools of mature and nascent transcripts respectively  <cit> .
although this represents an efficient approach, these protocols are time consuming and require high amounts of starting material.
they are therefore less suitable for studies based on tissue samples, where starting material is often a limiting factor.
in this study, we investigate the benefits of analyzing rna sequencing data from separated nuclear and cytosolic rna.
we have made improvements to an existing protocol for cell fractionation in order to more efficiently fractionate cytoplasmic and nuclear rnas from tissue samples.
we find that extraction of rna using our modified protocol results in pure subcellular rna populations with minimal levels of cross contamination.
rna-seq results from nuclear and cytosolic fractions are compared to polya+ and total rna-seq from the same tissue samples.
our results highlight significant advantages of performing rna-seq on cytosol and nuclear rnas, as compared to standard rna-seq protocols.
sequencing of nuclear rna provides insight into nascent transcript formation and processing, and cytosolic rna-seq leads to improved de novo assembly and splice junction detection.
RESULTS
cellular fractionation of cytoplasm and nucleus
to improve the efficiency of rna extraction from different subcellular fractions, the cytoplasmic and nuclear rna purification kit  was modified with the addition of a sucrose gradient and extra washing step .
the method for cellular fractionation of rna is outlined in figure  <dig>  our results show that the nuclear rna fractions are virtually free of ribosomal rna and that the cytoplasmic rna contained no traces of genomic dna .
the extraction takes less than  <dig>  hours and as little as 15 mg of tissue sample can be used as starting material, with no requirement for additional polya+ or chromatin purification kits.
in comparison, polya+ purification requires a larger amount of starting material  and takes a longer time to complete.
cytoplasmic and nuclear rna-seq
to investigate the separation and detection of mature transcripts from the cytoplasm and nascent transcripts from the nucleus, we purified cytoplasmic and nuclear rna from two human fetal frontal cortex tissue samples, denoted sample  <dig> and sample  <dig>  we then sequenced  the cytoplasmic and nuclear rna from both samples, along with total and polya+ rna from the same tissues.
while many genes have very clean peaks corresponding to the exons , we find that long genes with high expression levels show a surprisingly high intron read coverage in nuclear, total and polya+ rna.
conversely, the rna-seq coverage profiles revealed a striking enrichment of exonic reads in the cytoplasmic fraction compared to the other rna fractions .
these differences in intronic and exonic rna levels were validated using qrt-pcr by calculating amplification cycle number differences  between introns and exons in the cytoplasmic and polya+ rna fractions .
we used the raw  for calculations, without normalization, because house-keeping genes may not be equally represented in subcellular fractions as compared to total or polya+ selected rna.
however, similar results were obtained when normalizing against beta-actin .
in order to show the effect of the extra steps added to the commercial rna extraction protocol we also performed the same experiment on rna extracted using the kit without modifications .
in all experimentally validated genes, we found a higher ratio of exonic to intronic rna in the cytoplasmic rna compared with the polya+ fraction, demonstrating the efficiency of our protocol to enrich for mature rna transcripts .
unexpectedly, polya+ rnas showed high levels of intron coverage across entire transcripts, contradicting the idea that polya+ purification enriches exclusively for fully mature rnas.
to exclude issues with polya+ purification as a reason for this, we sequenced a high quality adult brain polya+ rna acquired from a commercial vendor .
since similar patterns were seen also for the commercial polya+ sample, the observed intronic coverage is not likely to be an artifact unique to the polya+ enrichment carried out in our laboratory.
in line with these findings, recent studies indicate that transcripts may be polyadenylated prior to the completion of splicing  <cit> .
this data, together with the biases associated with polya+ selection, may potentially provide an explanation for the high level of intronic rna in polya+ data.
comparing exonic-to-intronic enrichment between rna fractions
to quantify the relative enrichment of exonic reads compared to intronic on a global scale, we defined a ratio of exonic-to-intronic reads .
the ei-ratio is a number ranging from  <dig>  to  <dig> .
in the cytoplasmic rna, the ei-ratios were  <dig>  and  <dig>  in the two tissues.
for polya+ rna the ei-ratio ranged from  <dig> - <dig>  and even lower values were seen in total rna  and nuclear rna  .
as expected, these results show that intronic reads are present at high levels in the nuclear and total rna, but also highlight that there is a substantial fraction of intronic reads in polya+ rna.
importantly, our results demonstrate that cytoplasmic rna is significantly enriched for exons in comparison with all the other rna populations, implying that it is a preferable extraction method for studying completely processed mrna molecules.
on the other hand, if the aim is to study nascent transcripts, our results suggest that nuclear rna is the best choice since it gives the lowest ei-ratios .
interestingly, we observed lower ei-ratios for the polya+ and total rna fractions in sample  <dig> as compared to sample  <dig>  we explain this with biological differences in transcription levels between the two samples.
genes involved in the nervous system development often contain very long introns  <cit>  and our results indicate that these genes in sample  <dig>  which is from an earlier developmental stage, are transcribed at much higher levels, resulting in a higher fraction of intronic reads.
ei-ratios
for different rna fractions
cytoplasmic rna-seq improves the analysis of mature mrnas
to further evaluate the different rna fractions, we focused on the potential of our method to improve the detection and quantification of mature spliced transcripts.
given the higher ei-ratio in the cytoplasmic rna, we expect that it should be possible to identify a larger number of mature transcripts in rna-seq data from the cytoplasm compared with the other fractions.
we first investigated the ability to detect expressed transcripts in the different rna fractions, using the depth of coverage per million mapped reads  as a measure to quantify the expression levels of all exons in the human genome.
as expected, cytoplasmic rna-seq gives the highest dcpm levels for exons .
furthermore, by analyzing dcpm values at exonic positions compared with the background noise signal represented by the coverage on the anti-sense strand , we could estimate the number of expressed exons for each of the rna fractions .
in the cytoplasmic rna fraction we detect 8-19% more expressed exons than in polya+ rna and 29-49% more than in total rna, thereby corroborating a more efficient detection of exonic reads in the cytoplasmic fraction as compared to the polya+ or total rna fractions.
we used tophat  <cit>  to perform splice junction analysis on rna-seq data from sample  <dig>  after correcting for differences in number of total sequence reads, the cytosolic rna-seq were found to have around  <dig>   more junctions than polya+ rna-seq .
furthermore, the number of reads spanning splice junctions in the cytosolic rna-seq were roughly  <dig>   higher compared with the polya+ rna-seq .
these results confirm that the number of reads derived from spliced transcripts is greater in the cytosolic rna, and that each junction detected has better support in the cytosolic rna-seq data.
to evaluate the data in an unbiased way, without any prior information about gene coordinates, we then performed a de novo transcriptome assembly for each rna population using the trinity software  <cit> .
here, we analyzed sample  <dig> since it represents a more challenging dataset with smaller differences in ei-ratios between rna fractions.
the results show that the cytoplasmic rna fraction provides longer contigs, and featured 30% more transcripts longer than 1 kb compared with the polya+ fraction and almost  <dig> times more than in total rna .
this trend is consistent using a cutoff of 2 kb.
there were also more transcripts containing open reading frames  in the cytoplasmic fraction.
despite of the fact that our rna-seq data consists of short  and unpaired reads, which are not ideal for de novo assembly, our results clearly show that cytoplasmic rna gives a better transcriptome assembly compared with the other fractions.
shown are the n <dig> sizes , the total bases contained in contigs larger than  <dig> and  <dig> nucleotides and the number of transcripts with open reading frames  larger than  <dig> amino acids.
in all cases, the cytoplasmic assembly outperforms polya+ selection, as well as total rna.
trinity could not assemble the nuclear fraction due to long run times and we terminated the process after taking up  <dig>  cpu years.
nuclear rna-seq improves analysis of nascent transcription
the low ei-ratios in the nuclear rna fractions suggest that a high amount of nascent transcripts are being detected by nuclear rna sequencing.
to investigate this further we performed a global analysis of the sequence coverage across introns.
figure  <dig> shows the coverage for all four rna fractions and the commercial polya+ sample across introns of different lengths.
it has previously been observed that nascent transcripts give rise to a 5′-3′ negative gradient of rna-seq coverage across long introns  <cit> , and consistent with this we see such slopes in global analyses of long introns for the nuclear, total and polya+ fractions .
the 5′-3′ slope is associated with nascent transcript production and this pattern can also be used as an indicator of splicing dynamics  <cit> .
the steepest slopes are detected for the nuclear rna, indicating that it is the rna fraction containing the highest amount of nascent transcripts.
the second steepest slopes are found in total rna, followed by polya+ rna.
in contrast, the intronic coverage in the cytoplasmic rna-seq fraction is almost negligible and no slope at all is seen in the cytoplasmic rna.
for shorter introns the 5′-3′ gradients become less evident , and likely the reason for this is that the rna polymerase moves too quickly through the small introns to generate a detectable gradient of nascent rna.
our results thus show that the nuclear fraction contains the highest amount of nascent rnas, providing a more distinct 5′-3′ slope , indicating that sequencing of nuclear rna is preferable over total rna for studying ongoing transcription.
discussion
the advent of rna-seq has for the first time provided a method to examine the rna in cells and tissues in an unbiased way.
however, the methods of extracting rna differs and may give very different starting material for the rna-seq experiment.
it is important to understand the advantages and disadvantages of different extraction protocols, and the biases that are introduced in the rna preparation experiments.
there are now several rna protocols available for extracting specific classes of rna from the cell prior to sequencing.
these protocols are based on separation by size , type of rna or association with certain subcellular compartments or structures.
examples include polya+ selection, extraction of chromatin associated rna and extraction of cytosolic rna  <cit>  and rna polymerase associated rna  <cit> .
most data presented for subcellular fractions of rna are based on cell lines  <cit> .
here we present the improvement of a method for separation of cytosolic and nuclear rna and demonstrate that for many applications this is a more informative approach than sequencing polya+ and total rna.
in addition to the improved efficiency of this method to enrich for mature rna transcripts, cytoplasmic rna purification provides several technical advantages over conventional polya+ enrichment approaches.
the enrichment of polya+ rna directly from tissues and cell lines is a complicated and time-consuming procedure, requiring significant amounts of starting material.
cytoplasmic rna purification represents a more affordable and rapid protocol .
moreover, it requires only 15 mg of tissue to provide sufficient amounts of rna from both cytosol and nuclear fractions for rna-seq and subsequent validation experiments.
our approach has lower resolution for specific sub-classes of rna compared to more specialized molecular approaches such as gro-seq and net-seq, but has the benefit of providing a global picture of all rna in the cell, with increased resolution compared with conventional extraction protocols.
there are several potential biases when using polya+ rna, which may be avoided using the cytosolic fraction.
we find a surprisingly high fraction of intronic sequence reads in the polya+ rna-seq data.
although the results were better for the polya+ rna purchased from a commercial vendor, where a more stringent selection protocol was applied, we still find that > 50% of the intragenic reads map to introns.
these findings indicate that there may be significant unspecific capture of rna in the polya+ selection step, irrespective of the protocol used.
another explanation for the high intronic background in polya+ data is that some fraction of introns  is spliced only after the addition of the polya-tail  <cit> .
such a mechanism would explain why certain genes give a high intronic background in sequencing of polya-selected rna.
the coverage profiles across introns are clearly visible in figure  <dig>  where it is obvious that the polya+ baseline read coverage is significantly higher than in the rna extracted from the cytosol.
there have also been reports indicating that selection of mononucleotide stretches of adenines within nascent rna may create noise in polya+ seq data.
many of these biases are avoided by extracting only on the mature transcripts present in the cytosol.
the cytosolic rna contains high amounts of rrna that needs to be depleted prior to sequencing.
there are reports that rrna depletion may introduce 5′-3′ bias across transcripts  <cit> .
however, published data suggest that polya+ selected data show more 5′-3′ bias than does ribo-minus rna-seq data  <cit> .
if other biases are introduced by rrna depletion, biases in cytosolic rna-seq data would be similar to those in conventional total rna-seq, which also requires rrna depletion prior to sequencing.
compared to cytosolic rna, the nuclear fraction is less well suited for measuring mrna expression levels.
instead, the enrichment of nascent rna from the nuclear fraction is suitable for studying transcription dynamics without any further rrna depletion steps.
we show that signatures of co-transcriptional splicing are more distinct in the nuclear fraction than in total rna.
the level of intron coverage is also a good indicator of the rate of nascent transcript production, which does not always correlate with the level of processed mrna.
additionally, nuclear extraction is advantageous for studies of rna molecules that are primarily present in the nucleus, such as pri-microrna, rrna precursors and some long non-coding rnas  <cit> .
by extracting both cytosolic and nuclear rna fractions from the same tissue, it is also possible to study the relative abundance of the same transcripts in each fraction.
such analysis may provide insight into transcript processing, turnover and degradation.
for example, a high level of nascent transcription for a transcript found at relatively lower levels in the cytosol might be an indication of rapid cytosol turnover.
conversely, high levels a transcript in the cytosolic rna, combined with low nuclear levels for the same transcript may be an indication of high stability and long half-life for the mrna of that transcript.
CONCLUSIONS
in this paper, we report on the advantages of using rna-seq on separated cytosolic and nuclear rna, extracted using a modified and improved protocol.
analysis on separated nuclear and cytoplasmic fractions is valuable to study rna degradation patterns  and transport dynamics, intron retention patterns and mrna turnover.
our results show that extraction of nuclear rna is better than total rna for measuring of nascent transcript levels and for studies of mechanisms of splicing.
furthermore, we show that rna-sequencing of the cytoplasmic fraction shows an increased exonic coverage and minimal levels of intronic reads.
this results in significantly higher number of transcripts that can be assembled from this fraction when compared to total or polya+ preparations.
our data shows that sequencing of cytosolic rna yields substantially lower background from immature transcripts, and we propose that cytosolic rna-seq should be the method of choice for de novo transcriptome assembly and tissue specific expression profiling.

 the transcriptome is the complete catalogue of transcripts in the human cell. at any given time, a wide range of different rna molecules are present at different levels of maturation and processing. the rates and the dynamics of rna transcription and processing are unique for each cell. previous studies have highlighted the importance of creating a complete map of transcripts, and the importance of understanding how different physiological conditions, developmental stages and disease can affect expression and regulation. in the recent years, rna-sequencing has emerged as a standard procedure to study transcriptomes and measure levels of gene expression. studies using this method have significantly expanded our understanding of transcriptome complexity and provided new insights into the mechanisms of gene expression and transcriptional regulation in development and disease. however, many challenges still remain, primarily linked to data analysis and sample preparation. the starting material for rna-seq is typically total rna or polyadenylated rna, which both represent heterogeneous pools of rna molecules at different stages of maturation and processing. several limitations arise from analyzing these populations of rnas from whole cells. a commonly neglected problem is that total rna, but also polya+ rna to a lesser extent, contains substantial amounts of intronic rna originating from immature transcripts. this intronic background coverage is accentuated in long genes expressed at high levels, and is most noticeable in brain tissue where long neuronal genes tend to be highly expressed. the presence of intronic rnas may influence the sensitivity to detect transcripts, identify splice junctions and measure gene expression levels, as a large proportion of sequence reads is mapped to introns. also, oligo-dt purification steps are likely to introduce certain biases, such as unspecific retrieval of rna containing poly-a stretches within the transcribed sequence, 5′ to 3′ biases, or truncated transcripts resulting either from alternative polyadenylation signals within introns or rna degradation products. although total rna-seq has been shown to provide insight into ongoing transcription and co-transcriptional splicing in the nucleus, the simultaneous presence of mature rnas from the cytoplasm confounds the analysis of nuclear rna maturation steps. recently, two technologies, the genome-wide nuclear run-on sequencing and native elongating transcript sequencing, have been described to study nascent transcripts. gro-seq yields an overview of transcription dynamics and directionality by labeling transcriptionally engaged nascent transcripts genome-wide, followed by high-throughput sequencing. net-seq uses the stability of the ternary complex of dna, rna polymerase and nascent rna to capture and sequence nascent transcripts in living cells using endogenously expressed rnapii with a 3 × −flag epitope. these methods have successfully provided snapshots of ongoing transcription in cell lines. however, these methods do not provide any insight into posttranscriptional events, and are based upon manipulation of the normal physiological conditions of the cells and require extensive optimization and standardization. recent improvements in rna extraction protocols now make it possible to study specific pools of rna molecules, either by fractionation of subcellular compartments or by molecular capture of specific rna-associated targets. several kits for rna extraction from different cellular fractions are commercially available. however, these kits are associated with significant amounts of cross contamination between the fractions. to overcome the effects of cross contamination, recent studies used the selection of polya+ from the cytoplasmic fraction and chromatin-associated transcripts from the nuclear fraction to obtain more homogenous pools of mature and nascent transcripts respectively. although this represents an efficient approach, these protocols are time consuming and require high amounts of starting material. they are therefore less suitable for studies based on tissue samples, where starting material is often a limiting factor. in this study, we investigate the benefits of analyzing rna sequencing data from separated nuclear and cytosolic rna. we have made improvements to an existing protocol for cell fractionation in order to more efficiently fractionate cytoplasmic and nuclear rnas from tissue samples. we find that extraction of rna using our modified protocol results in pure subcellular rna populations with minimal levels of cross contamination. rna-seq results from nuclear and cytosolic fractions are compared to polya+ and total rna-seq from the same tissue samples. our results highlight significant advantages of performing rna-seq on cytosol and nuclear rnas, as compared to standard rna-seq protocols. sequencing of nuclear rna provides insight into nascent transcript formation and processing, and cytosolic rna-seq leads to improved de novo assembly and splice junction detection.  cellular fractionation of cytoplasm and nucleus to improve the efficiency of rna extraction from different subcellular fractions, the cytoplasmic and nuclear rna purification kit was modified with the addition of a sucrose gradient and extra washing step. the method for cellular fractionation of rna is outlined in figure  our results show that the nuclear rna fractions are virtually free of ribosomal rna and that the cytoplasmic rna contained no traces of genomic dna. the extraction takes less than  hours and as little as 15 mg of tissue sample can be used as starting material, with no requirement for additional polya+ or chromatin purification kits. in comparison, polya+ purification requires a larger amount of starting material and takes a longer time to complete. cytoplasmic and nuclear rna-seq to investigate the separation and detection of mature transcripts from the cytoplasm and nascent transcripts from the nucleus, we purified cytoplasmic and nuclear rna from two human fetal frontal cortex tissue samples, denoted sample and sample we then sequenced the cytoplasmic and nuclear rna from both samples, along with total and polya+ rna from the same tissues. while many genes have very clean peaks corresponding to the exons, we find that long genes with high expression levels show a surprisingly high intron read coverage in nuclear, total and polya+ rna. conversely, the rna-seq coverage profiles revealed a striking enrichment of exonic reads in the cytoplasmic fraction compared to the other rna fractions. these differences in intronic and exonic rna levels were validated using qrt-pcr by calculating amplification cycle number differences between introns and exons in the cytoplasmic and polya+ rna fractions. we used the raw for calculations, without normalization, because house-keeping genes may not be equally represented in subcellular fractions as compared to total or polya+ selected rna. however, similar results were obtained when normalizing against beta-actin. in order to show the effect of the extra steps added to the commercial rna extraction protocol we also performed the same experiment on rna extracted using the kit without modifications. in all experimentally validated genes, we found a higher ratio of exonic to intronic rna in the cytoplasmic rna compared with the polya+ fraction, demonstrating the efficiency of our protocol to enrich for mature rna transcripts. unexpectedly, polya+ rnas showed high levels of intron coverage across entire transcripts, contradicting the idea that polya+ purification enriches exclusively for fully mature rnas. to exclude issues with polya+ purification as a reason for this, we sequenced a high quality adult brain polya+ rna acquired from a commercial vendor. since similar patterns were seen also for the commercial polya+ sample, the observed intronic coverage is not likely to be an artifact unique to the polya+ enrichment carried out in our laboratory. in line with these findings, recent studies indicate that transcripts may be polyadenylated prior to the completion of splicing. this data, together with the biases associated with polya+ selection, may potentially provide an explanation for the high level of intronic rna in polya+ data. comparing exonic-to-intronic enrichment between rna fractions to quantify the relative enrichment of exonic reads compared to intronic on a global scale, we defined a ratio of exonic-to-intronic reads. the ei-ratio is a number ranging from to. in the cytoplasmic rna, the ei-ratios were and in the two tissues. for polya+ rna the ei-ratio ranged from - and even lower values were seen in total rna and nuclear rna. as expected, these results show that intronic reads are present at high levels in the nuclear and total rna, but also highlight that there is a substantial fraction of intronic reads in polya+ rna. importantly, our results demonstrate that cytoplasmic rna is significantly enriched for exons in comparison with all the other rna populations, implying that it is a preferable extraction method for studying completely processed mrna molecules. on the other hand, if the aim is to study nascent transcripts, our results suggest that nuclear rna is the best choice since it gives the lowest ei-ratios. interestingly, we observed lower ei-ratios for the polya+ and total rna fractions in sample as compared to sample we explain this with biological differences in transcription levels between the two samples. genes involved in the nervous system development often contain very long introns and our results indicate that these genes in sample which is from an earlier developmental stage, are transcribed at much higher levels, resulting in a higher fraction of intronic reads. ei-ratios for different rna fractions cytoplasmic rna-seq improves the analysis of mature mrnas to further evaluate the different rna fractions, we focused on the potential of our method to improve the detection and quantification of mature spliced transcripts. given the higher ei-ratio in the cytoplasmic rna, we expect that it should be possible to identify a larger number of mature transcripts in rna-seq data from the cytoplasm compared with the other fractions. we first investigated the ability to detect expressed transcripts in the different rna fractions, using the depth of coverage per million mapped reads as a measure to quantify the expression levels of all exons in the human genome. as expected, cytoplasmic rna-seq gives the highest dcpm levels for exons. furthermore, by analyzing dcpm values at exonic positions compared with the background noise signal represented by the coverage on the anti-sense strand, we could estimate the number of expressed exons for each of the rna fractions. in the cytoplasmic rna fraction we detect 8-19% more expressed exons than in polya+ rna and 29-49% more than in total rna, thereby corroborating a more efficient detection of exonic reads in the cytoplasmic fraction as compared to the polya+ or total rna fractions. we used tophat to perform splice junction analysis on rna-seq data from sample after correcting for differences in number of total sequence reads, the cytosolic rna-seq were found to have around more junctions than polya+ rna-seq. furthermore, the number of reads spanning splice junctions in the cytosolic rna-seq were roughly higher compared with the polya+ rna-seq. these results confirm that the number of reads derived from spliced transcripts is greater in the cytosolic rna, and that each junction detected has better support in the cytosolic rna-seq data. to evaluate the data in an unbiased way, without any prior information about gene coordinates, we then performed a de novo transcriptome assembly for each rna population using the trinity software. here, we analyzed sample since it represents a more challenging dataset with smaller differences in ei-ratios between rna fractions. the results show that the cytoplasmic rna fraction provides longer contigs, and featured 30% more transcripts longer than 1 kb compared with the polya+ fraction and almost times more than in total rna. this trend is consistent using a cutoff of 2 kb. there were also more transcripts containing open reading frames in the cytoplasmic fraction. despite of the fact that our rna-seq data consists of short and unpaired reads, which are not ideal for de novo assembly, our results clearly show that cytoplasmic rna gives a better transcriptome assembly compared with the other fractions. shown are the n sizes, the total bases contained in contigs larger than and nucleotides and the number of transcripts with open reading frames larger than amino acids. in all cases, the cytoplasmic assembly outperforms polya+ selection, as well as total rna. trinity could not assemble the nuclear fraction due to long run times and we terminated the process after taking up cpu years. nuclear rna-seq improves analysis of nascent transcription the low ei-ratios in the nuclear rna fractions suggest that a high amount of nascent transcripts are being detected by nuclear rna sequencing. to investigate this further we performed a global analysis of the sequence coverage across introns. figure  shows the coverage for all four rna fractions and the commercial polya+ sample across introns of different lengths. it has previously been observed that nascent transcripts give rise to a 5′-3′ negative gradient of rna-seq coverage across long introns, and consistent with this we see such slopes in global analyses of long introns for the nuclear, total and polya+ fractions. the 5′-3′ slope is associated with nascent transcript production and this pattern can also be used as an indicator of splicing dynamics. the steepest slopes are detected for the nuclear rna, indicating that it is the rna fraction containing the highest amount of nascent transcripts. the second steepest slopes are found in total rna, followed by polya+ rna. in contrast, the intronic coverage in the cytoplasmic rna-seq fraction is almost negligible and no slope at all is seen in the cytoplasmic rna. for shorter introns the 5′-3′ gradients become less evident, and likely the reason for this is that the rna polymerase moves too quickly through the small introns to generate a detectable gradient of nascent rna. our results thus show that the nuclear fraction contains the highest amount of nascent rnas, providing a more distinct 5′-3′ slope, indicating that sequencing of nuclear rna is preferable over total rna for studying ongoing transcription. discussion the advent of rna-seq has for the first time provided a method to examine the rna in cells and tissues in an unbiased way. however, the methods of extracting rna differs and may give very different starting material for the rna-seq experiment. it is important to understand the advantages and disadvantages of different extraction protocols, and the biases that are introduced in the rna preparation experiments. there are now several rna protocols available for extracting specific classes of rna from the cell prior to sequencing. these protocols are based on separation by size, type of rna or association with certain subcellular compartments or structures. examples include polya+ selection, extraction of chromatin associated rna and extraction of cytosolic rna and rna polymerase associated rna. most data presented for subcellular fractions of rna are based on cell lines. here we present the improvement of a method for separation of cytosolic and nuclear rna and demonstrate that for many applications this is a more informative approach than sequencing polya+ and total rna. in addition to the improved efficiency of this method to enrich for mature rna transcripts, cytoplasmic rna purification provides several technical advantages over conventional polya+ enrichment approaches. the enrichment of polya+ rna directly from tissues and cell lines is a complicated and time-consuming procedure, requiring significant amounts of starting material. cytoplasmic rna purification represents a more affordable and rapid protocol. moreover, it requires only 15 mg of tissue to provide sufficient amounts of rna from both cytosol and nuclear fractions for rna-seq and subsequent validation experiments. our approach has lower resolution for specific sub-classes of rna compared to more specialized molecular approaches such as gro-seq and net-seq, but has the benefit of providing a global picture of all rna in the cell, with increased resolution compared with conventional extraction protocols. there are several potential biases when using polya+ rna, which may be avoided using the cytosolic fraction. we find a surprisingly high fraction of intronic sequence reads in the polya+ rna-seq data. although the results were better for the polya+ rna purchased from a commercial vendor, where a more stringent selection protocol was applied, we still find that > 50% of the intragenic reads map to introns. these findings indicate that there may be significant unspecific capture of rna in the polya+ selection step, irrespective of the protocol used. another explanation for the high intronic background in polya+ data is that some fraction of introns is spliced only after the addition of the polya-tail. such a mechanism would explain why certain genes give a high intronic background in sequencing of polya-selected rna. the coverage profiles across introns are clearly visible in figure  where it is obvious that the polya+ baseline read coverage is significantly higher than in the rna extracted from the cytosol. there have also been reports indicating that selection of mononucleotide stretches of adenines within nascent rna may create noise in polya+ seq data. many of these biases are avoided by extracting only on the mature transcripts present in the cytosol. the cytosolic rna contains high amounts of rrna that needs to be depleted prior to sequencing. there are reports that rrna depletion may introduce 5′-3′ bias across transcripts. however, published data suggest that polya+ selected data show more 5′-3′ bias than does ribo-minus rna-seq data. if other biases are introduced by rrna depletion, biases in cytosolic rna-seq data would be similar to those in conventional total rna-seq, which also requires rrna depletion prior to sequencing. compared to cytosolic rna, the nuclear fraction is less well suited for measuring mrna expression levels. instead, the enrichment of nascent rna from the nuclear fraction is suitable for studying transcription dynamics without any further rrna depletion steps. we show that signatures of co-transcriptional splicing are more distinct in the nuclear fraction than in total rna. the level of intron coverage is also a good indicator of the rate of nascent transcript production, which does not always correlate with the level of processed mrna. additionally, nuclear extraction is advantageous for studies of rna molecules that are primarily present in the nucleus, such as pri-microrna, rrna precursors and some long non-coding rnas. by extracting both cytosolic and nuclear rna fractions from the same tissue, it is also possible to study the relative abundance of the same transcripts in each fraction. such analysis may provide insight into transcript processing, turnover and degradation. for example, a high level of nascent transcription for a transcript found at relatively lower levels in the cytosol might be an indication of rapid cytosol turnover. conversely, high levels a transcript in the cytosolic rna, combined with low nuclear levels for the same transcript may be an indication of high stability and long half-life for the mrna of that transcript.  in this paper, we report on the advantages of using rna-seq on separated cytosolic and nuclear rna, extracted using a modified and improved protocol. analysis on separated nuclear and cytoplasmic fractions is valuable to study rna degradation patterns and transport dynamics, intron retention patterns and mrna turnover. our results show that extraction of nuclear rna is better than total rna for measuring of nascent transcript levels and for studies of mechanisms of splicing. furthermore, we show that rna-sequencing of the cytoplasmic fraction shows an increased exonic coverage and minimal levels of intronic reads. this results in significantly higher number of transcripts that can be assembled from this fraction when compared to total or polya+ preparations. our data shows that sequencing of cytosolic rna yields substantially lower background from immature transcripts, and we propose that cytosolic rna-seq should be the method of choice for de novo transcriptome assembly and tissue specific expression profiling. 
both forms of rna represent heterogeneous pools of rna molecules at different levels of maturation and processing. we hypothesize that subcellular fractions of rna may provide a more accurate picture of gene expression. we present results for sequencing of cytoplasmic and nuclear rna after cellular fractionation of tissue samples. the starting material for rna sequencing studies is usually total rna or polya+ rna. conversely, the nuclear fraction shows an enrichment of unprocessed rna compared with total rna-seq, making it suitable for analysis of nascent transcripts and rna processing dynamics. our results show that cellular fractionation is a more rapid and cost effective approach than conventional polya+ enrichment when studying mature rnas. rna sequencingtranscriptomicsrna splicingrna purificationpolya+ selectioncytoplasmic rnanuclear rnanascent transcriptsde novo assemblytranscription profiling thus, rna-seq of separated cytosolic and nuclear rna can significantly improve the analysis of complex transcriptomes from mammalian tissues. in comparison with conventional polya+ rna, the cytoplasmic rna contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of rna-seq data. such heterogeneity, in addition to the biases associated with polya+ purification steps, may influence the analysis, sensitivity and the interpretation of rna-seq data.
 the starting material for rna sequencing studies is usually total rna or polya+ rna. both forms of rna represent heterogeneous pools of rna molecules at different levels of maturation and processing. such heterogeneity, in addition to the biases associated with polya+ purification steps, may influence the analysis, sensitivity and the interpretation of rna-seq data. we hypothesize that subcellular fractions of rna may provide a more accurate picture of gene expression.  we present results for sequencing of cytoplasmic and nuclear rna after cellular fractionation of tissue samples. in comparison with conventional polya+ rna, the cytoplasmic rna contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of rna-seq data. conversely, the nuclear fraction shows an enrichment of unprocessed rna compared with total rna-seq, making it suitable for analysis of nascent transcripts and rna processing dynamics.  our results show that cellular fractionation is a more rapid and cost effective approach than conventional polya+ enrichment when studying mature rnas. thus, rna-seq of separated cytosolic and nuclear rna can significantly improve the analysis of complex transcriptomes from mammalian tissues. rna sequencingtranscriptomicsrna splicingrna purificationpolya+ selectioncytoplasmic rnanuclear rnanascent transcriptsde novo assemblytranscription profiling 