DOI: 10.5061/dryad.cvdncjtcx
title: Data from: Automated workflow for the cell cycle analysis of (non-)adherent cells using a machine learning approach

description: Understanding the details of the cell cycle at the level of individual
 cells is critical for both cellular biology and cancer research. While
 existing methods using specific fluorescent markers have advanced our
 ability to study the cell cycle in cells that adhere to surfaces, there is
 a clear gap when it comes to non-adherent cells. In this study, we combine
 a specialized surface to improve cell attachment, the genetically-encoded
 FUCCI(CA)2 sensor, an automated image processing and analysis pipeline,
 and a custom machine-learning algorithm. This combined approach allowed us
 to precisely measure the duration of different cell cycle phases in
 non-adherent cells. Our method provided detailed information from hundreds
 of cells under different experimental conditions in a fully automated
 manner. We validated this approach in two different Acute Myeloid Leukemia
 (AML) cell lines, NB4 and Kasumi-1, which have unique cell cycle
 characteristics. Additionally, we tested the impact of drugs affecting the
 cell cycle in NB4 cells. Importantly, our cell cycle analysis system is
 freely available and has also been validated for use with adherent cells.
 In summary, this report introduces a comprehensive, automated method for
 studying the cell cycle in both adherent and non-adherent cells, offering
 a valuable tool for cancer research and drug development.
description: <strong>RNA extraction, RNA-seq protocol and data
 analysis</strong> Total RNA was extracted from
 dry pallets of cells collected prior- and post-acquisitions and purified
 using the Zymo Research Quick-RNA Miniprep (W/O directzol). Reverse
 transcription was performed with the SuperScript II Kit (Invitrogen),
 according to the manufacturer’s protocol. RNA-seq was performed according
 to the True-seq Low sample protocol selecting only polyadenylated
 transcripts. In brief, before starting mRNA isolation and library
 preparations the integrity of the total RNA was evaluated by running
 samples on a Bioanalyzer instrument by picoRNA Chip (Agilent), then
 converted into libraries of double stranded cDNA appropriate for next
 generation sequencing on the Illumina platform. The Illumina TruSeq v.2
 RNA Sample Preparation Kit was used following manufacturer’s
 recommendations. Briefly, 0.1-1 μg of total RNA were subjected to two
 rounds of mRNA purification by denaturing and letting the RNA bind to
 Poly‐T oligo-attached magnetic beads. Then fragmentation was performed
 exploiting divalent cations contained in the Illumina fragmentation buffer
 and high temperature. First and second strand cDNA is reverse transcribed
 from fragmented RNA using random hexamers. First strand cDNA was
 synthesized by SuperScript II (Invitrogen) reverse transcriptase and
 random primers and second strand cDNA synthesized by DNA polymerase I and
 Rnase H. The subsequent isolation of the cDNA was achieved by using AMPure
 XP beads (depending on the concentration used, these beads can efficiently
 recover PCR products of different sizes). The product recovered contained
 overhanging strands of various lengths due to the fragmentation procedure.
 The 5’ and 3’ ends of cDNA are repaired by the 3’-5’ exonuclease activity
 and the polymerase activity and adenylated at 3’ extremities before
 ligating specific Illumina oligonucleotides adapters followed by 15 cycles
 of PCR reaction using proprietary Illumina primers mix to enrich the DNA
 fragments. Prepared libraries were quality checked and quantified using
 Agilent high sensitivity DNA assay on a Bioanalizer 2100 instrument
 (Agilent Technologies). Raw reads 51bp PE for NB4 and Kasumi-1 cells were
 quality-filtered and aligned to the hg18 reference genome using
 nf-core/rnaseq v3.9 pipeline using STAR as aligner and Salmon for
 quantification with default parameters. Gene counts for each sample were
 log1p transformed, mean value among the two replicates was taken to
 compute Pearson correlation among gene expression pre- and post-
 time-lapse acquisition. <strong>EdU incorporation
 and assessment by Flow Cytometry</strong> A
 two-hour EdU pulse was performed by replacing half of the total media
 volume of the cells with 2X concentrated EdU in the corresponding growth
 medium, followed by subsequent fixation. Click-iT™ EdU Alexa Fluor™ 647
 Flow Cytometry Assay Kit (CN: C10419, Thermo-Fisher Scientific, Waltham,
 MA, USA) and Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa
 Fluor™ 647 dye (CN: C10340, Thermo-Fisher Scientific, Waltham, MA, USA)
 were used for flow cytometry and imaging, respectively. The experiments
 were performed according to the manufacturer’s protocols for the mentioned
 kits. DNA staining with DAPI using 500 μL of 5 μg/mL DAPI in PBS for
 10<sup>6</sup> cells, followed by overnight incubation at 4°C,
 was additionally performed for cell cycle profiling by flow cytometry.
 Alternatively, 5 μg/mL Hoechst® 33342 (Thermo-Fisher Scientific, Waltham,
 MA, USA) was used to stain DNA for imaging purposes.
 <strong>Fluorescence Time-Lapse
 Imaging</strong> Images were acquired with a
 Leica Thunder Imager (Leica Microsystems, Wetzlar, Germany), equipped with
 a Lumencor Spectra X Light Engine (Lumencor, Beaverton, USA) for
 fluorescence excitation, a motorized stage and a Leica DFC9000 GTC camera.
 For non-adherent cells, images were acquired with LAS X software (Leica
 Microsystems, Wetzlar, Germany, version 3.7.5.24914) using a 20X/0.75NA
 air objective and a binning 2x2 was applied to increase the SNR. The
 mCherry and mVenus signals were detected respectively with 540-580 nm and
 460-500 nm excitation filters, 585 and 505 nm dichroic mirrors and 592-668
 nm and 512-542 nm emission filters. The brightfield channel was also
 acquired for representation purposes. We imaged 20 to 25 fields of views
 per well and focal points were manually set in each position before
 starting the acquisition and kept constant during the whole time-lapse
 thanks to the Adaptive Focus Control (AFC, Leica Microsystems). The total
 duration of the time-lapse on non-adherent cells was 72 hours, and the
 time interval was set to 1 hour to prevent cell phototoxicity.
 The total duration of the time-lapse on MDA-MB-231 adherent cells
 was 120 hours, and the time interval was set to 30 minutes.
description: # Data from: Automated workflow for the cell cycle analysis of
 (non-)adherent cells using a machine learning approach
 [https://doi.org/10.5061/dryad.cvdncjtcx](https://doi.org/10.5061/dryad.cvdncjtcx) ## Description of the data and file structure #### File: salmon.merged.gene\_counts.tsv Total RNA was extracted from cell pellets collected and purified using the Zymo Research Quick-RNA Miniprep (W/O directzol). Reverse transcription was performed with the SuperScript II Kit (Invitrogen) per the manufacturer's protocol. RNA-seq was conducted following the TruSeq Low Sample protocol, selecting only polyadenylated transcripts. RNA integrity was assessed via Bioanalyzer (Agilent) before mRNA isolation and library preparation. The Illumina TruSeq v.2 RNA Sample Preparation Kit was used with 0.1-1 μg of RNA, undergoing two rounds of mRNA purification using Poly‐T oligo-attached magnetic beads. RNA was fragmented, and first- and second-strand cDNA synthesis was completed using SuperScript II and DNA polymerase I. AMPure XP beads were used to isolate cDNA. The 5’ and 3’ ends were repaired, adenylated, and ligated with Illumina adapters, followed by PCR enrichment. Libraries were quality checked and quantified with the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100. ### Files and variables #### File: salmon.merged.gene\_counts.tsv **Description:** Table express reads count for each gene in the pre- and post- acquisition samples in NB4 and Kasumi-1 cell line (obtained with Salmon from nf-core/rnaseq v3.9 pipeline) ##### Variables * gene_id: gene identifier * gene_name: name of the gene * Kasumi1.Post_rep1: reads count obtained in Kasumi-1 cells, first replica, after the time-lapse acquisition * Kasumi1.Post_rep2: reads count obtained in Kasumi-1 cells, second replica, after the time-lapse acquisition * Kasumi1.Pre_rep1: reads count obtained in Kasumi-1 cells, first replica, before the time-lapse acquisition * Kasumi1.Pre_rep2: reads count obtained in Kasumi-1 cells, first replica, before the time-lapse acquisition * NB4.Post_rep1: reads count obtained in NB4 cells, first replica, after the time-lapse acquisition * NB4.Post_rep2: reads count obtained in NB4 cells, second replica, after the time-lapse acquisition * NB4.Pre_rep1: reads count obtained in NB4 cells, first replica, before  the time-lapse acquisition * NB4.Pre_rep2: reads count obtained in NB4 cells, second replica, before the time-lapse acquisition ## Code/software Raw reads 51bp PE for NB4 and Kasumi-1 cells were quality-filtered and aligned to the hg18 reference genome using nf-core/rnaseq v3.9 pipeline using STAR as aligner and Salmon for quantification with default parameters. Gene counts for each sample were log1p transformed, mean value among the two replicates was taken to compute Pearson correlation among gene expression pre- and post- time-lapse acquisition. ## Description of the data and file structure ##### File: EdU\_.fcs\_files.zip This dataset contains FCS files from FACS experiments conducted on Kasumi-1 and MDA-MB-231 cells using FUCCI(CA)2 markers (mCherry and mVenus). Cells were stained with EdU Alexa Fluor™ 647 during a two-hour pulse and DAPI, as detailed in the methods section. The samples were acquired on a FACSAriaIII Fusion (FACSAriaIII) flow cytometer. The dataset includes 4 FCS files, representing both stained and unstained controls for each model. The gating strategy involved selecting live, single cells, followed by gates for PE-CF594-A :: mCherry, GFP-A :: mVenus, BV421-A :: DAPI, and APC-A :: EdU populations. The data is compatible with FlowJo and other FCS-compatible analysis software. #### Files and variables ##### File: EdU\_.fcs\_files.zip **Description:** The dataset includes 4 FCS files, representing both stained and unstained controls for each model. The gating strategy involved selecting live, single cells, followed by gates for PE-CF594-A :: mCherry, GFP-A :: mVenus, BV421-A :: DAPI, and APC-A :: EdU populations. ## Code/software The data is compatible with FlowJo and other FCS-compatible analysis software. ## Description of the data and file structure ##### files: 2022-04-22\_DMSO\_PALBO\_A1.zip,  2022-04-22\_DMSO\_PALBO\_A4.zip, TethisSTD\_A1.zip, TethisSTD\_A4.zip, TethisSTD\_C3.zip, ThetisSTD2\_A1.zip, ThetisSTD2\_A2.zip, ThetisSTD2\_B4.zip, ThetisSTD2\_C4.zip Approximately 250,000 NB4 or Kasumi-1 cells were plated in each well of the Tethis SBS 12-well multi-wells. NB4 were seeded in the presence of DMSO or 50nM of Palbociclib or 50nM of Ribociclib, or 50 nM of PF-06873600, in the culture medium at day 0. Kasumi-1 cells were seeded in culture medium. Images were acquired with a Leica Thunder Imager (Leica Microsystems, Wetzlar, Germany), equipped with a Lumencor Spectra X Light Engine (Lumencor, Beaverton, USA) for fluorescence excitation, a motorized stage and a Leica DFC9000 GTC camera. For non-adherent cells, images were acquired with LAS X software (Leica Microsystems, Wetzlar, Germany, version 3.7.5.24914) using a 20X/0.75NA air objective and a binning 2x2 was applied. The mCherry and mVenus signals were detected respectively with 540-580 nm and 460-500 nm excitation filters, 585 and 505 nm dichroic mirrors and 592-668 nm and 512-542 nm emission filters. The brightfield channel was also acquired for representation purposes. We imaged 20 to 25 fields of views per well and focal points were manually set in each position before starting the acquisition and kept constant during the whole time-lapse thanks to the Adaptive Focus Control (AFC, Leica Microsystems). The total duration of the time-lapse was 72 hours, and the time interval was set to 1 hour. #### Files and variables Compressed .lif files. ##### File: 2022-04-22\_DMSO\_PALBO\_A1.zip **Description:** NB4 cells treated with DMSO. Ch1=BF, Ch2=mVenus, Ch3=mCherry. Z=2. T=73. Regions=25. ##### File: 2022-04-22\_DMSO\_PALBO\_A4.zip **Description:** NB4 cells treated with Palbociclib 50 nM. Ch1=BF, Ch2=mVenus, Ch3=mCherry. Z=2. T=73. Regions=25. ##### File: TethisSTD\_A1.zip **Description:** NB4 cells treated with DMSO. Ch1=BF, Ch2=mVenus, Ch3=mCherry, Ch4=DRAQ7. Z=2. T=73. Regions=20. ##### File: TethisSTD\_A4.zip **Description:** NB4 cells treated with Palbociclib 50 nM. Ch1=BF, Ch2=mVenus, Ch3=mCherry, Ch4=DRAQ7. Z=2. T=73. Regions=20. ##### File: TethisSTD\_C3.zip **Description:** Kasumi-1cells untreated. Ch1=BF, Ch2=mVenus, Ch3=mCherry, Ch4=DRAQ7. Z=2. T=73. Regions=20. ##### File: ThetisSTD2\_A1.zip **Description:** NB4 cells treated with DMSO. Ch1=BF, Ch2=mVenus, Ch3=mCherry, Ch4=DRAQ7. Z=2. T=73. Regions=20. ##### File: ThetisSTD2\_A2.zip **Description:** NB4 cells treated with Ribociclib 50 nM. Ch1=BF, Ch2=mVenus, Ch3=mCherry, Ch4=DRAQ7. Z=2. T=73. Regions=20. ##### File: ThetisSTD2\_B4.zip **Description:** NB4 cells treated with PF 50 nM. Ch1=BF, Ch2=mVenus, Ch3=mCherry, Ch4=DRAQ7. Z=2. T=73. Regions=20. ##### File: ThetisSTD2\_C4.zip **Description:** NB4 cells treated with Palbociclib 50 nM. Ch1=BF, Ch2=mVenus, Ch3=mCherry, Ch4=DRAQ7. Z=2. T=73. Regions=20. ### Code/software This files can be opened, visualized and analysed with Fiji/ImageJ, after extraction. ## Description of the data and file structure #### **file: FUCCI\_adherent\_cells\_A1-R1.zip** One hundred thousand MDA-MB-231 cells expressing the FUCCI(CA)2 probe were plated in a Tethis SBS 12-well multi-well plate. Images were acquired with a Leica Thunder Imager (Leica Microsystems, Wetzlar, Germany). The mCherry and mVenus signals were detected respectively with 540-580 nm and 460-500 nm excitation filters, 585 and 505 nm dichroic mirrors and 592-668 nm and 512-542 nm emission filters. The brightfield channel was also acquired. Channel 1: BF Channel 2: mVenus Channel 3: mCherry Pixel size: 650 nm Z-step: 5.3 um time-frame: 30 min total duration: 120h ### Files and variables #### File: FUCCI\_adherent\_cells\_A1-R1.zip **Description:**  .lif file containing 3 channels (green, red and BF), 2 Z planes and 241 time frames.  ## Code/software This file can be opened with Fiji/ImageJ thanks to the Bioformats plug-in. ## Version changes: 2024-10-18: All datasets related to the manuscript have been added. Imaging, flow cytometry, and genomic data are now available under the same DOI.