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idr0056-study.txt
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# STUDY DESCRIPTION SECTION
# Section with generic information about the study including title, description, publication details (if applicable) and contact details
Comment[IDR Study Accession] idr0056
Study Title Identification of long noncoding RNAs in regulation of cell division
Study Type high content screen
Study Type Term Source REF EFO
Study Type Term Accession EFO_0007550
Study Description Long noncoding RNAs (lncRNAs) regulate many biological processes and have been implicated in development and disease pathogenesis. The exact function for the large majority of lncRNAs is still unknown. To address whether lncRNAs could play a role in cell division, we combined a microscopy-based high-content RNAi screen with dedicated image analysis workflows to identify lncRNAs that regulate mitotic progression, chromosome segregation and cytokinesis. By targeting over 2000 lncRNAs in HeLa cells, we discovered an assortment of new lncRNAs with unexplored functions in maintenance of genome stability.
Study Key Words long noncoding RNAs cell division mitosis cytokinesis chromosome segregeration
Study Organism Homo sapiens
Study Organism Term Source REF NCBITaxon
Study Organism Term Accession 9606
Study Screens Number 3
Study External URL
Study BioImage Archive Accession S-BIAD3
Study Public Release Date 2020-03-03
# Study Publication
Study PubMed ID 32296040
Study Publication Title A long noncoding RNA regulates microtubule behaviour during mitosis
Study Author List Stojic L, Lun ATL, Mascalchi P, Ernst C, Redmond AM, Mangei J, Cooper S, Barr AR, Taylor C, Franklin VNR, Bakal C, Marioni JC, Odom DT, Gergely F
Study PMC ID PMC7160116
Study DOI https://doi.org/10.1038/s41467-020-14978-7
# Study Contacts
Study Person Last Name Stojic
Study Person First Name Lovorka
Study Person Email lovorka.stojic@cruk.cam.ac.uk l.stojic@qmul.ac.uk
Study Person Address Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, United Kingdom
Study Person ORCID 0000-0001-6691-3396
Study Person Roles submitter
# Study License and Data DOI
Study License CC BY 4.0
Study License URL https://creativecommons.org/licenses/by/4.0/
Study Copyright Stojic et al
Study Data Publisher University of Dundee
Study Data DOI https://doi.org/10.17867/10000133
Term Source Name NCBITaxon EFO CMPO Fbbi
Term Source URI http://purl.obolibrary.org/obo/ http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/cmpo/ http://purl.obolibrary.org/obo/
# SCREEN SECTION
# Screen Section containing all information relative to each screen in the study including materials used, protocols names and description, phenotype names and description.
# For multiple screens this section should be repeated. Copy and paste the whole section below and fill out for the next screen.
Screen Number 1
Comment[IDR Screen Name] idr0056-stojic-lncrnas/screenA
Screen Data DOI https://doi.org/10.17867/10000133a
Screen Sample Type cell
Screen Description Hela cells were transfected with the human Lincode small interfering RNA (siRNA) library targeting 2231 lncRNAs. Following 48 hr incubation, cells were fixed and processed for immunostaining and subsequent automated image acquisition and analysis. Antibodies targeting CEP215 (to label centrosomes), alpha-tubulin (to label the microtubule cytoskeleton), phalloidin (to label the actin cytoskeleton) and Hoechst (to label nuclei) were used. 10x 384-well plates were used. The fixation and staining was carried out at the Institute for Cancer Research (ICR, London) using the PerkinElmer Cell:Explorer system coupled to automated liquid handling equipment. Solutions were dispensed using a Multidrop Combi Reagent dispenser (Thermo Fisher Scientific) and aspirated/washed using a Biotek washer with 96 pins. All plates were imaged using the PerkinElmer Opera high-content confocal screening platform with spinning disc. Thirty fields of view per well were captured using a 20x air objective, numerical aperture (NA) 0.45.
Screen Size Plates: 10 5D Images: Planes: Average Image Dimension (XYZCT): Total Tb:
Screen Example Images
Screen Imaging Method spinning disk confocal microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000253
Screen Technology Type RNAi screen
Screen Technology Term Source REF EFO
Screen Technology Term Accession EFO_0007551
Screen Type primary screen
Screen Type Term Source REF EFO
Screen Type Term Accession EFO_0007556
Screen Comments Screen A and B were used as two independent screens to identify new lncRNAs involved in regulation of cell division.
# Library section. The library file should be supplied separately and it should contain the reagents description including, at the absolute minimum: reagent ID, sequences and position in the layout (= plate + position in the plate)
Library File Name idr0056-screenA-annotation
Library File Format tab-delimited text
Library Type siRNA library
Library Type Term Source REF EFO
Library Type Term Accession EFO_0007564
Library Manufacturer Dharmacon
Library Version The design of this library is based on RefSeq version 54 and the siRNAs are arrayed as SMARTpools.
Library Experimental Conditions
Library Experimental Conditions Term Source REF Library Experimental Conditions Term Accession
Quality Control Description
# Protocols
Protocol Name growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007571 EFO_0007572 EFO_0007573
Protocol Description Growth Protocol - HeLa cells were grown in Dulbecco’s modified Eagle’s medium (Gibco, 41966-029) supplemented with 10% Fetal bovine serum (FBS, Thermo Fisher Scientific, 10500064). Exponentially growing cells were reverse transfected with 20nM of siRNA. An siRNA targeting exon 1 of lncRNA GNG12-AS1 (Silencer select, Life Technologies, S59962) and SMARTpool siRNAs targeting protein-coding gene CKAP5/Ch-TOG (GE Dharmacon, L-006847-00) were also included on each plate. CKAP5/Ch-TOG was used as a positive control as its depletion leads to mitotic delay and increased mitotic index. ECT2 SMARTpool siRNAs (GE Dharmacon, L-006450-00-0005) were used as a positive control only in the third validation screen as its depletion results in multinucleated cells. 48 hr after transfection cells were fixed and stained with appropriate antibodies.
Treatment Protocol - To redissolve the siRNA in the black 384-well PerkinElmer Cell Carrier plates (10 plates in total), 5 _l of OptiMEM medium (Thermo Fisher Scientific, 31985047) was added. The plates were centrifuged (1 min, 900g) and incubated at room temperature (RT) for 5 min. Lipofectamine RNAimax (Thermo Fisher Scientific, 13778150) was added in OptiMEM medium to a final concentration of 8 _l Lipofectamine to 1 ml OptiMEM medium and incubated at RT for 5 min. 5 _l of OptiMEM/Lipofectamine mix was then added to the plates. Plates were centrifuged and incubated at RT for 20 min. In the meantime, HeLa cells were trypinised and counted using an automated cell counter (Countess, Thermo Fisher Scientific). Cells were centrifuged at 1000g for 4 min, the medium was removed and the cells were resuspended in OptiMEM medium to a final concentration of 2000 cells/well. 10__l of cell suspension was added to the plates, plates were centrifuged and incubated at 37°C for 4 hours, before adding 10 _l of DMEM + 30% FBS + 3% P/S (penicillin/streptomycin, P/S) was added (final concentration 10% FBS, 1% P/S). Plates were centrifuged and incubated at 37°C for 48_hours before fixation. A Multidrop Combi Reagent dispenser (Thermo Fisher Scientific) was used throughout the transfection protocol to ensure even liquid addition.
HCS Library Protocol - For screen A, the plates were fixed by adding an equal volume of pre-warmed (37°C) 8% formaldehyde (Thermo Fischer Scientific, 28908)/PBS solution to the wells and incubated at 37°C for 10 min. The cells were permeabilised with pre-warmed PBS/0.2% Triton X-100 (Acros Organics, 327371000) for 15 min at RT. The cells were then blocked in 1% BSA/PBS for 1 hour at RT. To perform the immunostaining, the cells were incubated with primary antibodies against alpha-tubulin (Dm1alpha_, Sigma, TUB9026), CEP215/CDK5RAP272 and Alexa-Fluor® 568 Phalloidin (Thermo Fisher Scientific, A12380) for 2 hours at RT. The cells were washed three times in 1X PBS and incubated for 1 hour at RT with secondary antibodies Alexa Fluor® 488 (Thermo Fisher Scientific, A21206) and Alexa Fluor® 647 (Thermo Fisher Scientific, A31571). After three washes in 1X PBS, the cells were incubated with 1 µg/ml Hoechst (Sigma, H 33258, diluted in PBS) for 15_min at RT before a final wash in 1X PBS and imaging. For screen B, the same fixation protocol as described for screen A was used. For permeabilization, PBS/0.05% SDS was used for 20 min at RT. Blocking was performed as described above. For the immunostaining, cells were incubated with primary antibodies against gamma-tubulin (Sigma, GTU88) and phospho-histone H3 serine 10 (PHH3, Millipore, 06-570) for 2 hours at RT, washed three times with 1X PBS before incubation with secondary antibodies (Thermo Fisher Scientific, A-31571 and A-21206). After three 1X PBS washes, the cells were stained with alpha-tubulin (Serotec, MCA78G) and incubated with secondary antibody (Thermo Fisher Scientific, A-21434). All primary and secondary antibodies were diluted in 1% BSA/PBS. The third-pass validation screen was performed in two replicates with four technical replicates using the top 25 candidates from each of the categories (mitotic progression, cytokinesis) using the same antibodies as in screen B.
HCS Image Acquisition and Feature Extraction Protocol - All image analysis was performed using custom workflows created with the Columbus software (PerkinElmer). Several output parameters were evaluated from high content images: mitotic index (number of cells in mitosis), multinucleation index (number of multinucleated cells), number of viable cells, number of chromosome segregation errors (chromatin bridges and lagging chromatids) and number of cells with cytokinetic bridges .These are defined in more detail below: 1) Mitotic and multinucleation index: Nuclei were first segmented using Hoechst staining (which defines the total cell number). The false positives (e.g., dead cells) were discarded based on the nucleus area, alpha-tubulin and gamma-tubulin/CEP215 staining intensity. Multinucleated non-dividing cells were retained as a separate subpopulation using a two-step detection process: binucleated cells were isolated using size, aspect ratio and roundness parameters of close nucleus pairs. Other multinucleated cells were then identified among remaining cells for which alpha- or gamma-tubulin intensity was low in the perinuclear region. Further identification of mitotic cells/stages was accomplished using filters based on Hoechst for the nucleus shape and size, in combination with high PHH3 (screen B) or high alpha-tubulin and low CEP215 staining intensity (screen A). Notably, nuclei of cells in anaphase/telophase stage of mitosis were small, had elongated shape and exhibited low Hoechst integrated intensity (low amount of DNA among mitotic cells). The distance between both nuclei of cells in anaphase/telophase stage was the main criteria to discard two daughter non-mitotic cells (maximum of 0.65 µm and 2.6 µm, respectively). From all these sub-populations, we calculated mitotic and multinucleation index relative to the total number of live cells.
2) Chromosome segregation errors: We started from the previous identified subpopulation of cells in anaphase/telophase stage. We filtered cells according to alpha-tubulin staining intensity between nuclei, as cells in anaphase have lower alpha-tubulin intensity compared to the cells in telophase. This allowed us to identify only the cells in anaphase. To calculate the number of anaphase cells with chromosome segregation errors, the inter-nuclei space was used as the measuring area to calculate the remaining Hoechst signal. This captures both chromatin bridges and lagging chromatids.
3) Number of viable cells: The total number of viable cells was determined after removal of dead cells and cell debris with anaphase and telophase cells counted as one (despite exhibiting two nuclear segments). The same rule was applied for multinucleated cells.
4) Number of cytokinetic bridges: Cytokinetic bridges were defined as elongated high-intensity objects split into two parts that are positive for alpha-tubulin staining. We used prior segmentation of the cytoplasm and the Spot Finder feature to identify bridge half parts and sorted them as doublets by calculating distance between them. We discarded the false positive candidates based on the shape criteria and gamma-tubulin staining. The final number of cytokinetic bridges was divided by total number of viable cells.
To minimize the variation in the cell density between different wells among all ten 384-well plates, we divided the output numbers by the total number of cells per well (dead cells were not included). Multinucleated cells were considered to be single cells during counting. The ratios were then normalized between screen plates by calculating the average value per plate and finally the grand average of all ten plates, giving a reference mean ratio. Per-well ratios were scaled so that the per-plate average was equal to the reference. Z-scores (z) were calculated as follows for each parameter:
z= (x-µ)/σ where x represents the ratio for the feature of interest (e.g., mitotic/multinucleation index), µ represents the reference ratio and σ represents the standard deviation of ratios across wells.
# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession
# Raw Data Files
Raw Image Data Format
Raw Image Organization
# Feature Level Data Files (give individual file details unless there is one file per well) Feature Level Data File Name
Feature Level Data File Description
Feature Level Data File Format
Feature Level Data Column Name
Feature Level Data Column Type
Feature Level Data Column Description
# Processed Data Files
Processed Data File Name
Processed Data File Format tab-delimited text
Processed Data File Description
Processed Data Column Name
Processed Data Column Type
Processed Data Column Annotation Level
Processed Data Column Description
Processed Data Column Link To Library File
Screen Number 2
Comment[IDR Screen Name] idr0056-stojic-lncrnas/screenB
Screen Data DOI https://doi.org/10.17867/10000133b
Screen Sample Type cell
Screen Description Hela cells were transfecetd with the human Lincode small interfering RNA (siRNA) library targeting 2231 lncRNAs. Following 48 hr incubation, cells were fixed and processed for immunostaining and subsequent automated image acquisition and analysis. In screen B, phospho-histone H3 (PHH3; to specifically label mitotic cells), alpha-tubulin, gamma-tubulin (to label centrosomes) and Hoechst was used. 10x 384-well plates were used. The fixation and staining for both screen A and B was carried out at the Institute for Cancer Research (ICR, London) using the PerkinElmer Cell:Explorer system coupled to automated liquid handling equipment. Solutions were dispensed using a Multidrop Combi Reagent dispenser (Thermo Fisher Scientific) and aspirated/washed using a Biotek washer with 96 pins. All plates were imaged using the PerkinElmer Opera high-content confocal screening platform with spinning disc. Thirty fields of view per well were captured using a 20x air objective, numerical aperture (NA) 0.45.
Screen Size Plates: 10 5D Images: Planes: Average Image Dimension (XYZCT): Total Tb:
Screen Example Images
Screen Imaging Method spinning disk confocal microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000253
Screen Technology Type RNAi screen
Screen Technology Term Source REF EFO
Screen Technology Term Accession EFO_0007551
Screen Type primary screen
Screen Type Term Source REF EFO
Screen Type Term Accession EFO_0007556
Screen Comments Screen A and B were used as two independent screens to identify new lncRNAs involved in regulation of cell division.
# Library section. The library file should be supplied separately and it should contain the reagents description including, at the absolute minimum: reagent ID, sequences and position in the layout (= plate + position in the plate)
Library File Name idr0056-screenB-annotation
Library File Format tab-delimited text
Library Type siRNA library
Library Type Term Source REF EFO
Library Type Term Accession EFO_0007564
Library Manufacturer Dharmacon
Library Version The design of this library is based on RefSeq version 54 and the siRNAs are arrayed as SMARTpools.
Library Experimental Conditions
Library Experimental Conditions Term Source REF Library Experimental Conditions Term Accession
Quality Control Description
# Protocols
Protocol Name growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007571 EFO_0007572 EFO_0007573
Protocol Description Growth Protocol - HeLa cells were grown in Dulbecco’s modified Eagle’s medium (Gibco, 41966-029) supplemented with 10% Fetal bovine serum (FBS, Thermo Fisher Scientific, 10500064). Exponentially growing cells were reverse transfected with 20nM of siRNA. An siRNA targeting exon 1 of lncRNA GNG12-AS1 (Silencer select, Life Technologies, S59962) and SMARTpool siRNAs targeting protein-coding gene CKAP5/Ch-TOG (GE Dharmacon, L-006847-00) were also included on each plate. CKAP5/Ch-TOG was used as a positive control as its depletion leads to mitotic delay and increased mitotic index. ECT2 SMARTpool siRNAs (GE Dharmacon, L-006450-00-0005) were used as a positive control only in the third validation screen as its depletion results in multinucleated cells. 48 hr after transfection cells were fixed and stained with appropriate antibodies.
Treatment Protocol - To redissolve the siRNA in the black 384-well PerkinElmer Cell Carrier plates (10 plates in total), 5 _l of OptiMEM medium (Thermo Fisher Scientific, 31985047) was added. The plates were centrifuged (1 min, 900g) and incubated at room temperature (RT) for 5 min. Lipofectamine RNAimax (Thermo Fisher Scientific, 13778150) was added in OptiMEM medium to a final concentration of 8 _l Lipofectamine to 1 ml OptiMEM medium and incubated at RT for 5 min. 5 _l of OptiMEM/Lipofectamine mix was then added to the plates. Plates were centrifuged and incubated at RT for 20 min. In the meantime, HeLa cells were trypinised and counted using an automated cell counter (Countess, Thermo Fisher Scientific). Cells were centrifuged at 1000g for 4 min, the medium was removed and the cells were resuspended in OptiMEM medium to a final concentration of 2000 cells/well. 10__l of cell suspension was added to the plates, plates were centrifuged and incubated at 37°C for 4 hours, before adding 10 _l of DMEM + 30% FBS + 3% P/S (penicillin/streptomycin, P/S) was added (final concentration 10% FBS, 1% P/S). Plates were centrifuged and incubated at 37°C for 48_hours before fixation. A Multidrop Combi Reagent dispenser (Thermo Fisher Scientific) was used throughout the transfection protocol to ensure even liquid addition.
HCS Library Protocol - For screen A, the plates were fixed by adding an equal volume of pre-warmed (37°C) 8% formaldehyde (Thermo Fischer Scientific, 28908)/PBS solution to the wells and incubated at 37°C for 10 min. The cells were permeabilised with pre-warmed PBS/0.2% Triton X-100 (Acros Organics, 327371000) for 15 min at RT. The cells were then blocked in 1% BSA/PBS for 1 hour at RT. To perform the immunostaining, the cells were incubated with primary antibodies against alpha-tubulin (Dm1alpha_, Sigma, TUB9026), CEP215/CDK5RAP272 and Alexa-Fluor® 568 Phalloidin (Thermo Fisher Scientific, A12380) for 2 hours at RT. The cells were washed three times in 1X PBS and incubated for 1 hour at RT with secondary antibodies Alexa Fluor® 488 (Thermo Fisher Scientific, A21206) and Alexa Fluor® 647 (Thermo Fisher Scientific, A31571). After three washes in 1X PBS, the cells were incubated with 1 µg/ml Hoechst (Sigma, H 33258, diluted in PBS) for 15_min at RT before a final wash in 1X PBS and imaging. For screen B, the same fixation protocol as described for screen A was used. For permeabilization, PBS/0.05% SDS was used for 20 min at RT. Blocking was performed as described above. For the immunostaining, cells were incubated with primary antibodies against gamma-tubulin (Sigma, GTU88) and phospho-histone H3 serine 10 (PHH3, Millipore, 06-570) for 2 hours at RT, washed three times with 1X PBS before incubation with secondary antibodies (Thermo Fisher Scientific, A-31571 and A-21206). After three 1X PBS washes, the cells were stained with alpha-tubulin (Serotec, MCA78G) and incubated with secondary antibody (Thermo Fisher Scientific, A-21434). All primary and secondary antibodies were diluted in 1% BSA/PBS. The third-pass validation screen was performed in two replicates with four technical replicates using the top 25 candidates from each of the categories (mitotic progression, cytokinesis) using the same antibodies as in screen B.
HCS Image Acquisition and Feature Extraction Protocol - All image analysis was performed using custom workflows created with the Columbus software (PerkinElmer). Several output parameters were evaluated from high content images: mitotic index (number of cells in mitosis), multinucleation index (number of multinucleated cells), number of viable cells, number of chromosome segregation errors (chromatin bridges and lagging chromatids) and number of cells with cytokinetic bridges .These are defined in more detail below: 1) Mitotic and multinucleation index: Nuclei were first segmented using Hoechst staining (which defines the total cell number). The false positives (e.g., dead cells) were discarded based on the nucleus area, alpha-tubulin and gamma-tubulin/CEP215 staining intensity. Multinucleated non-dividing cells were retained as a separate subpopulation using a two-step detection process: binucleated cells were isolated using size, aspect ratio and roundness parameters of close nucleus pairs. Other multinucleated cells were then identified among remaining cells for which alpha- or gamma-tubulin intensity was low in the perinuclear region. Further identification of mitotic cells/stages was accomplished using filters based on Hoechst for the nucleus shape and size, in combination with high PHH3 (screen B) or high alpha-tubulin and low CEP215 staining intensity (screen A). Notably, nuclei of cells in anaphase/telophase stage of mitosis were small, had elongated shape and exhibited low Hoechst integrated intensity (low amount of DNA among mitotic cells). The distance between both nuclei of cells in anaphase/telophase stage was the main criteria to discard two daughter non-mitotic cells (maximum of 0.65 µm and 2.6 µm, respectively). From all these sub-populations, we calculated mitotic and multinucleation index relative to the total number of live cells.
2) Chromosome segregation errors: We started from the previous identified subpopulation of cells in anaphase/telophase stage. We filtered cells according to alpha-tubulin staining intensity between nuclei, as cells in anaphase have lower alpha-tubulin intensity compared to the cells in telophase. This allowed us to identify only the cells in anaphase. To calculate the number of anaphase cells with chromosome segregation errors, the inter-nuclei space was used as the measuring area to calculate the remaining Hoechst signal. This captures both chromatin bridges and lagging chromatids.
3) Number of viable cells: The total number of viable cells was determined after removal of dead cells and cell debris with anaphase and telophase cells counted as one (despite exhibiting two nuclear segments). The same rule was applied for multinucleated cells.
4) Number of cytokinetic bridges: Cytokinetic bridges were defined as elongated high-intensity objects split into two parts that are positive for alpha-tubulin staining. We used prior segmentation of the cytoplasm and the Spot Finder feature to identify bridge half parts and sorted them as doublets by calculating distance between them. We discarded the false positive candidates based on the shape criteria and gamma-tubulin staining. The final number of cytokinetic bridges was divided by total number of viable cells.
To minimize the variation in the cell density between different wells among all ten 384-well plates, we divided the output numbers by the total number of cells per well (dead cells were not included). Multinucleated cells were considered to be single cells during counting. The ratios were then normalized between screen plates by calculating the average value per plate and finally the grand average of all ten plates, giving a reference mean ratio. Per-well ratios were scaled so that the per-plate average was equal to the reference. Z-scores (z) were calculated as follows for each parameter:
z= (x-µ)/σ where x represents the ratio for the feature of interest (e.g., mitotic/multinucleation index), µ represents the reference ratio and σ represents the standard deviation of ratios across wells.
# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession
# Raw Data Files
Raw Image Data Format
Raw Image Organization
# Feature Level Data Files (give individual file details unless there is one file per well) Feature Level Data File Name
Feature Level Data File Description
Feature Level Data File Format
Feature Level Data Column Name
Feature Level Data Column Type
Feature Level Data Column Description
# Processed Data Files
Processed Data File Name
Processed Data File Format tab-delimited text
Processed Data File Description
Processed Data Column Name
Processed Data Column Type
Processed Data Column Annotation Level
Processed Data Column Description
Processed Data Column Link To Library File
Screen Number 3
Comment[IDR Screen Name] idr0056-stojic-lncrnas/screenC
Screen Data DOI https://doi.org/10.17867/10000133c
Screen Sample Type cell
Screen Description The third-pass validation screen was performed in two replicates with four technical replicates using the top 25 candidates from each of the categories (mitotic progression, cytokinesis) using phospho-histone H3 (PHH3; to specifically label mitotic cells), alpha-tubulin (to label the microtubule cytoskeleton), gamma-tubulin (to label centrosomes) and Hoechst (DNA). Correlation coefficients between replicate plates in third screen were calculated by Spearman’s rank correlation (mitotic index = 0.967888, viability = 0.93249, multinucleation index = 0.995897, cytokinetic bridges = 0.898324). The fixation and staining for the validation screen was carried out at the Institute for Cancer Research (ICR, London) using the PerkinElmer Cell:Explorer system coupled to automated liquid handling equipment. Solutions were dispensed using a Multidrop Combi Reagent dispenser (Thermo Fisher Scientific) and aspirated/washed using a Biotek washer with 96 pins. All plates were imaged using the PerkinElmer Opera high-content confocal screening platform with spinning disc. Thirty fields of view per well were captured using a 20x air objective, numerical aperture (NA) 0.45.
Screen Size Plates: 10 5D Images: Planes: Average Image Dimension (XYZCT): Total Tb:
Screen Example Images
Screen Imaging Method spinning disk confocal microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000253
Screen Technology Type RNAi screen
Screen Technology Term Source REF EFO
Screen Technology Term Accession EFO_0007551
Screen Type Validation
Screen Type Term Source REF EFO
Screen Type Term Accession EFO_0007558
Screen Comments Validation screen was used to confirm lcnRNA candidates involved in mitotic progression and cytokinesis.
# Library section. The library file should be supplied separately and it should contain the reagents description including, at the absolute minimum: reagent ID, sequences and position in the layout (= plate + position in the plate)
Library File Name idr0056-screenC-annotation
Library File Format tab-delimited text
Library Type siRNA library
Library Type Term Source REF EFO
Library Type Term Accession EFO_0007564
Library Manufacturer Dharmacon
Library Version The design of this library is based on RefSeq version 54 and the siRNAs are arrayed as SMARTpools beside GNG12-AS1 (single siRNA targeting exon1 of GNG12-AS1; Stojic et al Nature Communications 2016). Library Experimental Conditions
Library Experimental Conditions Term Source REF Library Experimental Conditions Term Accession
Quality Control Description
# Protocols
Protocol Name growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007571 EFO_0007572 EFO_0007573
Protocol Description Growth Protocol - HeLa cells were grown in Dulbecco’s modified Eagle’s medium (Gibco, 41966-029) supplemented with 10% Fetal bovine serum (FBS, Thermo Fisher Scientific, 10500064). Exponentially growing cells were reverse transfected with 20nM of siRNA. An siRNA targeting exon 1 of lncRNA GNG12-AS1 (Silencer select, Life Technologies, S59962) and SMARTpool siRNAs targeting protein-coding gene CKAP5/Ch-TOG (GE Dharmacon, L-006847-00) were also included on each plate. CKAP5/Ch-TOG was used as a positive control as its depletion leads to mitotic delay and increased mitotic index. ECT2 SMARTpool siRNAs (GE Dharmacon, L-006450-00-0005) were used as a positive control only in the third validation screen as its depletion results in multinucleated cells. 48 hr after transfection cells were fixed and stained with appropriate antibodies.
Treatment Protocol - To redissolve the siRNA in the black 384-well PerkinElmer Cell Carrier plates (10 plates in total), 5 _l of OptiMEM medium (Thermo Fisher Scientific, 31985047) was added. The plates were centrifuged (1 min, 900g) and incubated at room temperature (RT) for 5 min. Lipofectamine RNAimax (Thermo Fisher Scientific, 13778150) was added in OptiMEM medium to a final concentration of 8 _l Lipofectamine to 1 ml OptiMEM medium and incubated at RT for 5 min. 5 _l of OptiMEM/Lipofectamine mix was then added to the plates. Plates were centrifuged and incubated at RT for 20 min. In the meantime, HeLa cells were trypinised and counted using an automated cell counter (Countess, Thermo Fisher Scientific). Cells were centrifuged at 1000g for 4 min, the medium was removed and the cells were resuspended in OptiMEM medium to a final concentration of 2000 cells/well. 10__l of cell suspension was added to the plates, plates were centrifuged and incubated at 37°C for 4 hours, before adding 10 _l of DMEM + 30% FBS + 3% P/S (penicillin/streptomycin, P/S) was added (final concentration 10% FBS, 1% P/S). Plates were centrifuged and incubated at 37°C for 48_hours before fixation. A Multidrop Combi Reagent dispenser (Thermo Fisher Scientific) was used throughout the transfection protocol to ensure even liquid addition.
HCS Library Protocol - For screen A, the plates were fixed by adding an equal volume of pre-warmed (37°C) 8% formaldehyde (Thermo Fischer Scientific, 28908)/PBS solution to the wells and incubated at 37°C for 10 min. The cells were permeabilised with pre-warmed PBS/0.2% Triton X-100 (Acros Organics, 327371000) for 15 min at RT. The cells were then blocked in 1% BSA/PBS for 1 hour at RT. To perform the immunostaining, the cells were incubated with primary antibodies against alpha-tubulin (Dm1alpha_, Sigma, TUB9026), CEP215/CDK5RAP272 and Alexa-Fluor® 568 Phalloidin (Thermo Fisher Scientific, A12380) for 2 hours at RT. The cells were washed three times in 1X PBS and incubated for 1 hour at RT with secondary antibodies Alexa Fluor® 488 (Thermo Fisher Scientific, A21206) and Alexa Fluor® 647 (Thermo Fisher Scientific, A31571). After three washes in 1X PBS, the cells were incubated with 1 µg/ml Hoechst (Sigma, H 33258, diluted in PBS) for 15_min at RT before a final wash in 1X PBS and imaging. For screen B, the same fixation protocol as described for screen A was used. For permeabilization, PBS/0.05% SDS was used for 20 min at RT. Blocking was performed as described above. For the immunostaining, cells were incubated with primary antibodies against gamma-tubulin (Sigma, GTU88) and phospho-histone H3 serine 10 (PHH3, Millipore, 06-570) for 2 hours at RT, washed three times with 1X PBS before incubation with secondary antibodies (Thermo Fisher Scientific, A-31571 and A-21206). After three 1X PBS washes, the cells were stained with alpha-tubulin (Serotec, MCA78G) and incubated with secondary antibody (Thermo Fisher Scientific, A-21434). All primary and secondary antibodies were diluted in 1% BSA/PBS. The third-pass validation screen was performed in two replicates with four technical replicates using the top 25 candidates from each of the categories (mitotic progression, cytokinesis) using the same antibodies as in screen B.
HCS Image Acquisition and Feature Extraction Protocol - All image analysis was performed using custom workflows created with the Columbus software (PerkinElmer). Several output parameters were evaluated from high content images: mitotic index (number of cells in mitosis), multinucleation index (number of multinucleated cells), number of viable cells, number of chromosome segregation errors (chromatin bridges and lagging chromatids) and number of cells with cytokinetic bridges .These are defined in more detail below: 1) Mitotic and multinucleation index: Nuclei were first segmented using Hoechst staining (which defines the total cell number). The false positives (e.g., dead cells) were discarded based on the nucleus area, alpha-tubulin and gamma-tubulin/CEP215 staining intensity. Multinucleated non-dividing cells were retained as a separate subpopulation using a two-step detection process: binucleated cells were isolated using size, aspect ratio and roundness parameters of close nucleus pairs. Other multinucleated cells were then identified among remaining cells for which alpha- or gamma-tubulin intensity was low in the perinuclear region. Further identification of mitotic cells/stages was accomplished using filters based on Hoechst for the nucleus shape and size, in combination with high PHH3 (screen B) or high alpha-tubulin and low CEP215 staining intensity (screen A). Notably, nuclei of cells in anaphase/telophase stage of mitosis were small, had elongated shape and exhibited low Hoechst integrated intensity (low amount of DNA among mitotic cells). The distance between both nuclei of cells in anaphase/telophase stage was the main criteria to discard two daughter non-mitotic cells (maximum of 0.65 µm and 2.6 µm, respectively). From all these sub-populations, we calculated mitotic and multinucleation index relative to the total number of live cells.
2) Chromosome segregation errors: We started from the previous identified subpopulation of cells in anaphase/telophase stage. We filtered cells according to alpha-tubulin staining intensity between nuclei, as cells in anaphase have lower alpha-tubulin intensity compared to the cells in telophase. This allowed us to identify only the cells in anaphase. To calculate the number of anaphase cells with chromosome segregation errors, the inter-nuclei space was used as the measuring area to calculate the remaining Hoechst signal. This captures both chromatin bridges and lagging chromatids.
3) Number of viable cells: The total number of viable cells was determined after removal of dead cells and cell debris with anaphase and telophase cells counted as one (despite exhibiting two nuclear segments). The same rule was applied for multinucleated cells.
4) Number of cytokinetic bridges: Cytokinetic bridges were defined as elongated high-intensity objects split into two parts that are positive for alpha-tubulin staining. We used prior segmentation of the cytoplasm and the Spot Finder feature to identify bridge half parts and sorted them as doublets by calculating distance between them. We discarded the false positive candidates based on the shape criteria and gamma-tubulin staining. The final number of cytokinetic bridges was divided by total number of viable cells.
To minimize the variation in the cell density between different wells among all ten 384-well plates, we divided the output numbers by the total number of cells per well (dead cells were not included). Multinucleated cells were considered to be single cells during counting. The ratios were then normalized between screen plates by calculating the average value per plate and finally the grand average of all ten plates, giving a reference mean ratio. Per-well ratios were scaled so that the per-plate average was equal to the reference. Z-scores (z) were calculated as follows for each parameter:
z= (x-µ)/σ where x represents the ratio for the feature of interest (e.g., mitotic/multinucleation index), µ represents the reference ratio and σ represents the standard deviation of ratios across wells.
# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession
# Raw Data Files
Raw Image Data Format
Raw Image Organization
# Feature Level Data Files (give individual file details unless there is one file per well) Feature Level Data File Name
Feature Level Data File Description
Feature Level Data File Format
Feature Level Data Column Name
Feature Level Data Column Type
Feature Level Data Column Description
# Processed Data Files
Processed Data File Name
Processed Data File Format tab-delimited text
Processed Data File Description
Processed Data Column Name
Processed Data Column Type
Processed Data Column Annotation Level
Processed Data Column Description
Processed Data Column Link To Library File