EU-RNA-sequencing (EU-RNA-seq) maps primary transcripts with high coverage (fig. S3B)
because reads span introns and exons of annotated transcripts and are largely absent from intergenic regions (Fig. 1, B and C, and fig. S3C),
with reproducibility (fig. S3D). The distribution
of asynchronous FPKMs (fragments per kilo-base of transcript model per million fragments
mapped) (fig. S4A) and wide dynamic range helps
distinguish genes that can reliably be detected
(FPKM ≥ 19) (Fig. 1C and fig. S4B) from those
that cannot (FPKM < 19) (fig. S4C). Reads from
nonspecific RNA, not transcribed during the pulse
(“NoEU”), primarily mapped to exons of highly
abundant, stable mRNAs, such as for ALB (fig.
S4D), and were removed from all samples without
affecting asynchronous FPKMs as compared with
microarray data (fig. S4E). We conclude that EU-RNA-seq is a robust and reliable method for
mapping the nascent transcriptome.
With three spike-in replicates, we observed 8074
transcripts (3689 genes) (fig. S5A) consistently
expressed in mitosis (fig. S5B and table S5). The
mean decrement in expression was fivefold, with
a much narrower range in expression compared
with that in asynchronous cells (Fig. 1D). Of the
mitotic transcripts, 97% are expressed above 5%
of their asynchronous level (fig. S5C), and the different relative rank expression profiles (fig. S5D)
indicate that the mitotic transcriptome is distinct from that in asynchronous cells. Furthermore,
3329 mitotically expressed genes are expressed
higher in mitosis than can be attributed to the ~3%
contaminating asynchronous cells, based on co-alignment of the mitotic and asynchronous reads
with those from 222 adult human liver RNA-seq
studies (fig. S5E and table S6). Thus, the low-level
transcription seen in the mitotic population cannot
be explained by contaminating interphase cells.
We quantified fluorescein isothiocyanate–
uridine 5′-triphosphate (FITC-UTP) incorporation in mitotic HUH7 cells with or without the
transcriptional inhibitor a-amanitin, which has
detected transcription at centromeres ( 22). Nascent RNA signals were evident across chromosome
0 40 80105 165 300min
interphase mitosis interphase mitosis
FPKM = 19
Fig. 1. EU-RNA-seq and direct FITC-UTP labeling reveal extensive transcription in mitosis.
(A) Pulse-labeling during mitosis and mitotic exit. (B) Reads span exons and introns, not intergenic
regions. y axis, fragments per million fragments mapped (FPM). (C) A representative transcript
with an FPKM of 19. (D) FPKMs of mitotically expressed transcripts, in mitosis and in asynchronous
cells. Bar, mean; whiskers, quartiles; P < 0.001, n = 8074 transcripts. (E to G) Interphase or
(H to M) mitotic cells labeled with FITC-UTP; white boxes are magnified in (K) to (M).
(N to P) Interphase or (Q to V) mitotic cells treated with a-amanitin and labeled with FITC-UTP;
white boxes are magnified in (Q) to (S). Arrow, RNA signal; arrowhead, no RNA signal.
number of unique genes
DAPI introns overlay
n = 484
0 0 20 40 60
Fig. 2. Markedly active genes in mitosis. Naturally occurring mitotic cells
in an asynchronous population stained for (A, E, and I) 4′,6-diamidino-2-
phenylindole and (B, F, and J) exonic and (C, G, and K) intronic RNA.
(D, H, and L) Colocalization at primary transcripts. White arrows, exon;
yellow arrows, intron. (M) FPKMs of mitotically enriched genes in mitotic and
asynchronous cells. Bar, mean; whiskers, quartiles; P < 0.001, n = 484 genes.
(N) Representative GO categories for mitotically enriched genes.