Protocols from the manuscript

Cell Culture, Synchronization and RNA preparation

HeLa S3 cells were plated (2x10⁶ cells) in 150 mm tissue culture dishes in Dulbecco's Modified Eagle's Medium with 10% Fetal Bovine Serum and 100U penicillin-streptomycin (Invitrogen Life Technologies, Carlsbad CA).  Cells were arrested in S phase using a double thymidine block or in mitosis with a thymidine-nocodazole block essentially as described previously (Whitfield et al., 2000) and in the supplemental material.  Poly (A) RNA was prepared from cells collected at intervals (typically 1-2 hrs) by lysing cells directly on the plate using the Fast Track 2.0 mRNA isolation kit (Invitrogen Life Technologies, Carlsbad CA).  Synchrony was monitored by flow cytometry analysis of propidium iodide stained cells (Stanford Shared FACS facility).

Mitotic cells were collected every 10 minutes using an automated cell shaker (Eliassen et al., 1998), stored on ice and plated at two-hour intervals in fresh pre-warmed media (at least 10⁶ cells for each time point).  Since only a small number of cells can be obtained by mitotic shake-off, total RNA was prepared using ULTRASPEC RNA isolation system (BIOTECX, Houston TX).  The number of cells in S phase at each point was determined using a 5-Bromo-2'-deoxyuridine (BrdU) Labeling and Detection Kit I (Roche Molecular Biochemicals, Indianapolis IN).

Reference RNA was prepared from asynchronously growing HeLa cells using Trizol (Invitrogen Life Technologies, Carlsbad CA) and poly(A) RNA isolated by affinity chromatography on oligo-dT cellulose (Amersham Pharmacia Biotech, Piscataway NJ).  Poly(A) RNA was used as a reference in all experiments except the mitotic shake-off, where total RNA was labeled.

cDNA synthesis and microarray hybridization

RNA from synchronous cells was reverse-transcribed into Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway NJ) labeled cDNA and reference RNA reverse-transcribed into Cy3-dUTP (Amersham Pharmacia Biotech, Piscataway NJ) labeled cDNA using standard methods (Eisen and Brown, 1999) (Details are available at http://genome-www.stanford.edu/Human-CellCycle/Hela/).  Total RNA samples from cells collected in the mitotic shake-off experiment and total reference RNA were first amplified using a modified Eberwine protocol prior to cDNA synthesis (Wang et al., 2000), then labeled cDNA was prepared from the amplified RNA.

Spotted cDNA microarrays, containing 22,692 elements representing approximately 16,332 different human genes or containing 43,198 elements representing approximately 29,621 genes (estimated by Unigene Clusters), were manufactured in the Stanford Microarray Facility (http://www.microarray.org).  Equal amounts of Cy5- and Cy3-labeled cDNA were hybridized to spotted cDNA microarrays and scanned using a GenePix 4000A Scanner (Axon Instruments, Union City CA).  Detailed protocols are available at http://brownlab.stanford.edu/protocols.html.

Data processing

Data were extracted by superimposing a grid over each array using GenePix 3.0 software (Axon Instruments, Union City CA).  Spots of poor quality, determined by visual inspection, were removed from further analysis.  Data collected for each array were stored in the Stanford Microarray Database (SMD) and are available from SMD at http://genome-www.stanford.edu/microarray/ (Sherlock et al., 2001).

Only features with signal intensity at least 20% above background in both Cy-5 and Cy-3 channels and for which adequate quality data were obtained for at least 80% of the samples in a given time course were analyzed further.  Data points that did not meet these criteria are blank in the primary data tables.  Log₂(Cy5/Cy3) was retrieved for each data point and used for all analysis, where (Cy5/Cy3) is the normalized ratio of the background corrected intensities, as defined in SMD (Sherlock et al., 2001).

Because of systematic differences between experiments (e.g. array batch, labeling methods and synchronization methods) each time course was centered independently by filtering out the first, most significant eigengene (Alter et al., 2000), which was a dominant, constant vector.  Since SVD requires a full data matrix, missing data points were estimated using a k-nearest neighbors algorithm (Troyanskaya et al., 2001) with k=12.  These imputed values were used throughout the analysis and but were restored to “unknown” status in the figures and left blank in the primary data tables (http://genome-www.stanford.edu/Human-CellCycle/Hela/).

Identification of periodically expressed transcripts

A Fourier Transform (Eq. 1 – 3) was applied to the data for each clone in an experiment (Spellman et al., 1998) and the resulting vector (C, eq. 3) of the sine (A) and cosine (B) coefficients was stored, where T is the cell cycle period, t is the time after release, f is the phase offset and ratio(t) is the normalized Cy-5/Cy-3 expression ratio at time t.  The value of f was initially set to zero.  The values obtained for C were determined over a range of 40 values of T equally spaced 1hr above and below the estimated cell cycle period and the resulting values averaged and stored.

The optimal cell cycle period was determined by finding the value of T where the largest numbers of genes pass an arbitrary magnitude cutoff (D, eq. 4).  Fourier transforms were applied to the data series for each gene (eq. 1-3), with equally spaced values of T, from 0 to 40 hrs in 15-minute increments.  The number of genes whose magnitude (D, eq. 4) exceeded the arbitrary cut-off of 3, 5, or 7 was plotted and a period (T) was chosen that maximized the numbers of genes exceeding our arbitrary thresholds.  In most cases, the determined value of T was consistent with the data obtained by flow cytometry.

Because each experiment does not start at exactly the same point in the cell cycle, an offset (f, Eq 1-2) was calculated for each dataset relative to the first double thymidine arrest.  The magnitudes from the Fourier transform (D, eq. 4) for the 1000 highest scoring clones were summed using different values of f, equally spaced between 0 and 2p.  The offset that gave the highest average combined magnitude (D, eq. 4) between the two datasets for these 1000 genes was then used.  The Fourier transform was then repeated on the remaining datasets with the following values of T and f: Thy-Thy 2 (T = 15.5 hrs, f = 0.5 rad), Thy-Thy 3 (T = 15.4, f = 0.0 rad), Thy-Noc (T = 18.5, f = 3.2 rad), and mitotic selection (T = 24.5, f = 3.5 rad).  The vectors C (eq. 3) for all 5 datasets were then summed and the genes ranked according to the magnitude (D, eq. 4) of their combined vectors.  Note, the Thy-Noc and mitotic shake-off experiments, which arrest cells in mitosis, have offsets of approximately half a cycle (p radians) from Thy-Thy 1, which arrest cells at G1/S.

Since the gene expression profiles of many cell cycle genes do not precisely match sine and cosine curves, the expression profile of each gene was correlated to an idealized vector obtained from known genes expressed in each cell cycle phase (G1/S, S, G2, G2/M) as defined in Figure 2A.  Using a standard Pearson correlation, each gene received a peak correlation score defined as the highest absolute value correlation between one of the four idealized vectors and its expression profile (Spellman et al., 1998).  The absolute value of the peak correlation was used to scale the magnitude of the vector (C, eq. 2) generating a “periodicity score” for each gene (Table 1).

To estimate the minimum periodicity score for a cell cycle regulated gene, the above analysis was repeated on randomized data.  The data were randomized either within rows only, or within both rows and columns, for each of the five datasets starting with the imputed, SVD centered data.  The Fourier transform and correlations were applied using the previously calculated values of T and f; the resulting vectors (C, eq. 3) were combined for each dataset.  The magnitude (D, eq. 4) of the Fourier transform was scaled by each “gene’s” peak correlation to one of the four ideal expression profiles.  This analysis, including the data randomization, was repeated ten times and the scores combined by averaging the score for each of the highest scoring “genes” from each randomization, followed by the second highest, third highest etc.  The estimated false positive rate at a given periodicity score is the number of “genes” that obtain at least that score in the randomized data.  We chose a minimum periodicity score of 3.29, which gave us 1333 clones at an initial false positive estimate of 1% when the data was randomized in rows.  Repeating this analysis 10 times gave an estimated 10 false positives (0.75 %; periodicity scores of 5.18 - 3.33) when the data were randomized only within rows and two false positives (0.15 %; periodicity scores of 3.72 - 3.30) when the data were randomized in both rows and columns.

The false positive estimate, calculated above, is likely to underestimate the true false positive rate because it does not take into account genes that received a high Fourier scores because they exhibited a sinusoidal pattern in only part of a time course.  To filter out genes that did not show periodic expression, autocorrelations for each 1333 clones were calculated (Eq. 5).  The autocorrelation A is equal to the summation over all times t of the product of the ratio at t multiplied by the ratio at a time t + T, where T is the cell cycle period determined by Fourier analysis.  If the data for a gene repeats with a period T, the autocorrelation will be high.

Autocorrelation scores were calculated for Thy-Thy 3 and the Thy-Noc experiments because they represent multiple cell cycles and because points were taken at equally spaced intervals throughout the time course.  In experiment 3 autocorrelations were calculated for periods (T, eq. 5) of 15, 16 and 17 hours.  In experiment 4 autocorrelations were calculated for periods of 16, 18 and 20 hours.  The score for each gene in a given time course was taken to be the maximum of the three autocorrelations.  The final autocorrelation score assigned to each gene was the sum of the scores calculated for each of the two time courses.

Autocorrelations were used as a filter to remove genes that showed transient expression despite receiving a high periodicity score. 199 genes with a negative autocorrelation (a negative autocorrelation indicates the measured ratios do not repeat every cell cycle) were eliminated from the initial set of putative cell cycle regulated genes.  Autocorrelations were also calculated for data randomized in rows, whereupon few genes received negative autocorrelations in the randomized data indicating that the negative scores are unlikely to occur by chance. The distribution of autocorrelation scores is shown in Supplemental Figure 16

Our final list contains 1134 clones that correspond to 874 UNIGENE clusters (UNIGENE build 143, released 11-09-2001, 21 clones not found in UNIGENE, 66 map to more than one UNIGENE cluster).  The data for all 1134 clones as well as the primary data are available at http://genome-www.stanford.edu/Human-CellCycle/Hela/

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Supplemental Protocols not in the manuscript

This supplement provides experimental details of cell synchronization and array hybridization.  Detailed protocols are also available from http://brownlab.stanford.edu/protocols.html

Cell Synchronization

HeLa S3 cells were plated at a density of 2x10⁶ cells in 150 mm tissue culture dishes in Dulbecco's Modified Eagle's Medium (Invitrogen Life Technologies, Carlsbad CA) with 10% Fetal Bovine Serum (Invitrogen Life Technologies, Carlsbad CA) and 100U penicillin-streptomycin (Invitrogen Life Technologies, Carlsbad CA).  Cells were arrested in S phase by a double thymidine block as has previously been described (Whitfield et al., 2000).  24 hrs after plating cells were blocked with 2 mM thymidine for 17-18 hrs, released from the arrest for 9 hrs and arrested a second time with thymidine.  After 18 hrs, the cells were released and followed for 30 – 46 hrs depending on the experiment (see Figures 1-2).  To obtain populations of cells in mitosis, cells were arrested in 2mM thymidine for 17-18 hrs, released for 4 hrs and blocked in 100 ng/ml nocodazole for 12 hrs.  Floating mitotic cells were collected, washed twice in 1X PBS and replated at a density of 4x10⁶ cells in each 150 cm dish.  The cultures were typically at 10 – 25% confluence at the synchronous release and reached confluence by the end of the time course.  A detailed protocol for synchronization of HeLa cells by double thymidine or thymidine-nocodazole blocks is available at http://genome-www.stanford.edu/Human-CellCycle/Hela/mandm.shtml.

cDNA synthesis and microarray hybridization.

RNA from synchronous cells was reverse-transcribed into Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway NJ) labeled cDNA and reference RNA was reverse transcribed into Cy3-dUTP (Amersham Pharmacia Biotech, Piscataway NJ) labeled cDNA using Superscript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad CA).  Since only a small number of cells could be obtained from mitotic shake-off, total RNA was first amplified using a modified Eberwine protocol prior to cDNA synthesis (Wang et al., 2000); total reference RNA was amplified in parallel.  cDNA synthesis was primed using either oligo-dT and random hexamer (for the first and second double thymidine time courses; Thy-Thy1 and Thy-Thy2), oligo-dT alone (in third double thymidine time course and the thymidine-nocodazole time course; Thy-Thy2 and Thy-Noc), or with random hexamer alone (for mitotic shake-off).

Equal amounts of Cy5- and Cy3-labeled cDNA were mixed and applied to spotted cDNA microarrays in 3X SSC/0.1% SDS in a final volume of 28 mL (24K arrays) or 38 mL (48K arrays) under 22x40 or 22x60 cm coverslips (Corning or Fisher Brand).  Hybridizations were performed overnight (typically 12 – 18 hrs) in custom hybridization chambers (Monterey Industries, Richmond CA) at 65^oC.  After hybridization, arrays were washed for 2 minutes in each of four different wash solutions (2X SSC/0.1% SDS, 2X SSC, 1XSSC and 0.1X SSC) and excess wash solution removed by centrifugation at 500xg for 5 minutes.  All arrays were scanned using a GenePix 4000A Scanner (Axon Instruments, Union City CA).  Detailed protocols for microarray manufacture, RNA isolation, cDNA synthesis and microarray hybridization are available at http://brownlab.stanford.edu/protocols.html.