DOI: 10.5061/dryad.tqjq2bw93
title: Endpoint gastruloid-on-microraft analysis (EGOMRA)

description: Although adherent two-dimensional human gastruloids are highly
 reproducible and provide insights into early embryogenesis, no available
 automated technology can screen and sort large numbers of these
 near-millimeter-sized complex structures for large-scale assays.
 Consequently, we developed a microraft array-based technology to perform
 image-based assays of large numbers of fixed or living gastruloids and
 sort individual gastruloids for downstream assays, such as gene expression
 analysis. We created datasets with metrics describing the microraft arrays
 and the templates used to make the arrays, micropatterning of
 extracellular matrix (ECM) to form gastruloids, gastruloid formation, and
 gastruloids formed on both multi-well plates and microraft arrays. The
 microraft array platform was used to assay individual euploid and
 aneuploid (possessing abnormal numbers of chromosomes) gastruloids with
 clear phenotypic and gene expression differences. Both noggin (NOG) and
 keratin 7 (KRT7), two genes involved in spatial patterning within
 gastruloids, were upregulated in aneuploid relative to that in the euploid
 gastruloids. Moreover, relative NOG and KRT7 expressions were negatively
 correlated with DNA/area. The microraft arrays will empower novel screens
 of single gastruloids for a better understanding of key mechanisms
 underlying phenotypic differences between gastruloids.
description: # Endpoint gastruloid-on-microraft analysis (EGOMRA) This repository hosts
 datasets describing metrics for microraft arrays, micropatterning, and
 two-dimensional human gastruloid culture for the following manuscript in
 APL Bioengineering: Development of Large Scale Gastruloid Array to
 Identify Aberrant Developmental Phenotypes. We submitted raw data
 (EGOMRA.zip) and images of both fixed/living gastruloids on microrafts
 (EGOMRA Images.zip). ## **Files and variables** ### **File: EGOMRA.zip**
 #### **Automated Microraft Image Analysis Metrics** Metrics describing the
 accuracy of automated grading of gastruloid placement on microrafts. #####
 ***Automated Microraft Image Analysis Metrics.csv*** Array Type: fixed or
 living gastruloid array Replicate: replicate number Positive: number of
 identified centered gastruloids Negative: number of identified not
 centered gastruloids False Positive: number of falsely identified centered
 gastruloids False Negative: number of falsely identified not centered
 gastruloids True Positive: number of correctly identified centered
 gastruloids True Negative: number of correctly identified not centered
 gastruloids Positive: number of true centered gastruloids Negative: number
 of true not centered gastruloids Sensitivity: percentage of correctly
 identified out of true centered gastruloids Specificity: percentage of
 correctly identified out of true not centered gastruloids #####
 ***Automated Microraft Image Analysis Metrics\_Means.csv*** Array Type:
 fixed or living gastruloid array Summary: mean or standard deviation
 (stdev) across replicates for array type Sensitivity: percentage of
 correctly identified out of true centered gastruloids Specificity:
 percentage of correctly identified out of true not centered gastruloids
 #### **Automated Microraft Imaging Times** Time durations of array
 imaging. ##### ***Automated Microraft Imaging Times.csv*** Array Type:
 fixed or living gastruloid array Num Channels: total number of channels
 Replicate: replicate number Scan_Time (s): time to scan entire array in s
 Scan_Time (min): time to scan entire array in min Scan_Time Per Channel
 (min): time to scan entire array per channel in min ##### ***Automated
 Microraft Imaging Times\_Means.csv*** Array Type: fixed or living
 gastruloid array Num Channels: total number of channels Summary: mean or
 standard deviation (stdev) across replicates Scan_Time (min): time to scan
 entire array in min Scan_Time Per Channel (min): time to scan entire array
 per channel in min #### **Automated Microraft Isolation Successes**
 Success rates of microraft release & collection. ##### ***Automated
 Microraft Isolation Successes.csv*** Variable: microraft release or
 collection Replicate/Summary: replicate number or summary [mean or
 standard deviation (stdev)] across replicates Count: number of microrafts
 attempted to be isolated Success %: percent success rate #### **Automated
 Microraft Isolation Times** Time durations of microraft isolations. #####
 ***Automated Microraft Isolation Times.csv*** Replicate: replicate number
 FindRaftTime: time to locate target microraft in s Count: number of needle
 actuations required to release microraft from array NeedleTimes_{actuation
 number}: time for single needle actuation in s CollectionTimes: time to
 collect and deposit microraft in collection plate in s SingleRaftTimes:
 total time to locate, release, and collect each microraft in s #####
 ***Automated Microraft Isolation Times\_Means.csv*** Variable:
 FindRaftTimes, NeedleTimes, CollectionTimes, SingleRaftTimes (see above)
 Replicate/Summary: replicate number or summary [mean or standard deviation
 (stdev)] across replicates Value: value of variable #### **Bead
 Distribution** Area and distance to microraft edge of superparamagnetic
 beads within individual microrafts. ##### ***Bead Distribution.csv***
 Replicate: replicate number ArrayLoc: array location (middle or
 peripheral) Raft: microraft number RaftArea: area of microraft in px^2
 RaftCen: pixel coordinates (x, y) of microraft centroid within image
 BeadArea: area of beads in px^2 NormBeads: area of beads normalized by
 area of microraft FromEdge_Mean: mean distance of bead pixels to microraft
 edge in px FromEdge_Stdev: standard deviation of distance of bead pixels
 to microraft edge in px ##### ***Bead Distribution\_Means.csv***
 Replicate: replicate number or across all replicates (all) ArrayLoc: array
 location (middle or peripheral) NormBead_Mean: mean normalized area of
 beads NormBead_Stdev: standard deviation for normalized area of beads
 FromEdge_Mean: mean distance of bead pixels to microraft edge in px
 FromEdge_Stdev: standard deviation for distance of bead pixels to
 microraft edge in px #### **Fixed Gastruloid Fluor Analysis** Features
 extracted from high-quality fixed gastruloids. ##### ***Fixed Gastruloid
 Fluor Analysis.csv*** Replicate: replicate number Raft: microraft number
 Variable: ThreshVal_{channel} [intensity threshold value used in
 segmentation]; FArea_{channel} [fluorescence area of segmented region in
 px^2]; NormFArea_{channel} [fluorescence area of segmented region
 normalized to area of gastruloid]; Eccen_{channel} [eccentricity of
 segmented region]; Radius_{channel} [radius of segmented region in px]
 Value: value of variable ##### ***Fixed Gastruloid Fluor
 Analysis\_Means.csv*** Replicate: replicate number or across all
 replicates (all) Count: sample size (number of microrafts for
 replicate-level means or number of replicates) Variable:
 ThreshVal_{channel} [intensity threshold value used in segmentation];
 FArea_{channel} [fluorescence area of segmented region in px^2];
 NormFArea_{channel} [fluorescence area of segmented region normalized to
 area of gastruloid]; Eccen_{channel} [eccentricity of segmented region];
 Radius_{channel} [radius of segmented region in px] Mean: replicate-level
 mean or mean across replicates Stdev: replicate-level standard deviation
 or standard deviation across replicates #### **Living Gastruloid Fluor
 Analysis** Features extracted from high-quality living gastruloids. #####
 ***Living Gastruloid Fluor Analysis.csv*** Replicate: replicate number
 Condition: euploid or aneuploid Raft: microraft number Variable: Eccen
 [eccentricity of calcein stain]; Farea_Hull [fluorescence area of calcein
 stain in px^2]; NormDNA [fluorescence area of Hoechst 33342 stain
 normalized by fluorescence area of calcein stain]; Radius [radius of
 calcein stain in px] Value: value of variable ##### ***Living Gastruloid
 Fluor Analysis\_Means.csv*** Replicate: replicate number or across all
 replicates (all) Condition: euploid or aneuploid Count: sample size
 (number of microrafts for replicate-level means or number of replicates)
 Variable: Eccen [eccentricity of calcein stain]; FArea_Hull [fluorescence
 area of calcein stain in px^2]; NormDNA [fluorescence area of Hoechst
 33342 stain normalized by fluorescence area of calcein stain]; Radius
 [radius of calcein stain in px] Mean: replicate-level mean or mean across
 replicates Stdev: replicate-level standard deviation or standard deviation
 across replicates SEM: standard error across replicates (only calculated
 across replicates, NaN otherwise) #### **Living Gastruloid NOG & KRT7
 qPCR** Measurements and calculations of noggin (NOG) and keratin 7 (KRT7)
 gene expression via qPCR. “Living Gastruloid NOG & KRT7
 qPCR_Filtered.csv” contains manually filtered data (outlier GAPDH CT
 values and CT values exceeding 38 were excluded from furth analysis) from
 “Living Gastruloid NOG & KRT7 qPCR.csv”. ##### ***Living Gastruloid
 NOG & KRT7 qPCR.csv*** Plate: individual plates for NOG and KRT7
 assays (both plates contained GAPDH assays) Well Position: well positions
 for each sample ``` Sample Name: {replicate number}_{condition: euploid
 (Eu) or aneuploid (An)}_{sample number} ``` Target Name: gene expression
 assay target (GAPDH, NOG, KRT7) CT: cycle threshold ##### ***Living
 Gastruloid NOG & KRT7 qPCR\_Filtered.csv*** Plate: individual plates
 for NOG and KRT7 assays (both plates contained GAPDH assays) Well
 Position: well positions for each sample ``` Sample Name: {replicate
 number}_{condition: euploid (Eu) or aneuploid (An)}_{sample number} ```
 Target Name: gene expression assay target (GAPDH, NOG, KRT7) CT: cycle
 threshold ##### ***Living Gastruloid NOG & KRT7
 qPCR\_RelativeExpression.csv*** ``` Samp_Name: {replicate
 number}_{condition: euploid (Eu) or aneuploid (An)}_{sample number} ```
 Replicate: replicate number Condition: euploid (Eu) or aneuploid (An)
 Samp_Num: sample number Target: gene expression assay target (NOG or KRT7)
 dCT: delta cycle threshold (CT) calculated by subtracting CT by average CT
 value for GAPDH Average dCT: average dCT for euploid condition (control)
 across sample numbers for same replicate ddCT: delta dCT calculated by
 subtracting each dCT from “Average dCT” 2^-ddCT: calculated for each ddCT
 value to determine relative expression level (each cycle is two-fold
 difference) NormDNA: normalized DNA content (fluorescence area of Hoechst
 stain divided by total area of gastruloid) ##### ***Living Gastruloid NOG
 & KRT7 qPCR\_RelativeExpression\_Means.csv*** Replicate: replicate
 number or across all replicates (all) Condition: euploid (Eu) or aneuploid
 (An) Target: gene expression assay target (NOG or KRT7) Mean:
 replicate-level mean or mean across replicates for relative expression
 level (2^-ddCT) Stdev: replicate-level standard deviation or standard
 deviation across replicates for relative expression level (2^-ddCT) ####
 **Microraft Dimensions** Measurements of microraft dimensions (edge
 length, flatness, thickness). ##### ***Microraft Dimensions\_Edge.csv***
 Replicate: replicate number ArrayLoc: array location (middle or
 peripheral) Raft: microraft number Edge: edge number EdgeLen: edge length
 in µm ##### ***Microraft Dimensions\_Flatness.csv*** Replicate: replicate
 number ArrayLoc: array location (middle or peripheral) Raft: microraft
 number RaftLen: microraft edge length from cross-section view in µm
 FlatLen: length of flat portion of microraft from cross-section view in µm
 FlatProp: flat proportion calculated by dividing “FlatLen” by “RaftLen”
 ##### ***Microraft Dimensions\_Thickness.csv*** Replicate: replicate
 number ArrayLoc: array location (middle or peripheral) Raft: microraft
 number Orthogonal: cross-sectional view [XZ (1) or YZ (2)] RaftLoc:
 microraft location (center or edge) Thickness: thickness in µm #####
 ***Microraft Dimensions\_Means.csv*** Variable: Edge [edge length in µm];
 Flatness [length of flat portion of microraft divided by total microraft
 edge length]; Thickness [thickness of microraft in µm] Replicate:
 replicate number or across all replicates (all) ArrayLoc: array location
 (middle, peripheral, Total) Raft: microraft number or across all
 microrafts (all) RaftLoc: microraft location (center or edge) only for
 thickness, NaN otherwise Mean: replicate-level mean or mean across
 replicates Stdev: replicate-level standard deviation or standard deviation
 across replicates #### **Microraft Grading Count** Numbers of microrafts
 graded as centered [centered gastruloid on microraft], missing [missing
 microraft], empty [empty microraft], or border [gastruloid contacting
 border of microraft]. ##### ***Microraft Grading Count.csv*** Replicate:
 replicate number ArrType: array type (fixed or living) Grade: microraft
 grade (see above) ArrLoc: array location (middle, peripheral, total)
 Count: number of microrafts for each grade PercRaft: percentage of
 microrafts for each grade (out of 529 microrafts per replicate) #####
 ***Microraft Grading Count\_Means.csv*** Grade: microraft grade (see
 above) ArrLoc: array location (middle, peripheral, total) Mean: mean
 percentage of microrafts for each grade (out of 529 microrafts per
 replicate) Stdev: standard deviation for percentage of microrafts for each
 grade #### **Patterning Metrics** Metrics describing AF647-labeled
 Matrigel micropatterns on microrafts. ##### ***Patterning
 Metrics\_Diameter.csv*** Replicate: replicate number ArrayLoc: array
 location (middle or peripheral) Pattern: micropattern number Diameter:
 diameter of micropattern in µm ##### ***Patterning
 Metrics\_Diameter\_Means.csv*** Replicate: replicate number or across all
 replicates (all) ArrayLoc: array location (middle, peripheral, total) Mean
 Diameter: replicate-level mean or mean across replicates for micropattern
 diameter in µm Stdev Diameter: replicate-level standard deviation or
 standard deviation across replicates for micropattern diameter in µm SEM
 Diameter: replicate-level standard error or standard error across
 replicates for micropattern diameter in µm (only calculated for across
 replicates, NaN otherwise) Mean Radius: replicate-level mean or mean
 across replicates for micropattern radius in µm (only calculated for
 across replicates, NaN otherwise) Stdev Radius: replicate-level standard
 deviation or standard deviation across replicates for micropattern radius
 in µm (only calculated for across replicates, NaN otherwise) SEM Radius:
 replicate-level standard error or standard error across replicates for
 micropattern radius in µm  (only calculated for across replicates, NaN
 otherwise) ##### ***Patterning Metrics\_FromCenter.csv*** Replicate:
 replicate number ArrayLoc: array location (middle or peripheral) Pattern:
 micropattern number Distance: distance along line drawn across widest part
 of micropattern in µm Intensity: intensity value in arbitrary units along
 line drawn across widest part of micropattern ##### ***Patterning
 Metrics\_FromCenter\_Smooth.csv*** *Empty cells because of rolling average
 calculation (50 sliding window size).* Replicate: replicate number
 ArrayLoc: array location (middle or peripheral) Pattern: micropattern
 number Distance: distance along line drawn across widest part of
 micropattern in µm Intensity: intensity value in arbitrary units along
 line drawn across widest part of micropattern CenDist: half-length of line
 drawn across widest part of micropattern in µm FromCenDist: distance from
 the center of line drawn across widest part of micropattern in µm CenInt:
 average intensity within 10 µm of micropattern center NormInt: normalized
 intensity by dividing intensity value by “CenInt” for each micropattern
 NormInt_Smooth: “NormInt” smoothed via rolling average (50 sliding window
 size) ##### ***Patterning Metrics\_Grading.csv*** Replicate: replicate
 number Grade: micropattern grade [Centered (centered micropattern within
 microraft), Missing (missing micropattern), Border (micropattern
 contacting microraft border)] ArrayLoc: array location (middle,
 peripheral, total) Count: number of micropatterns for each grade PerRaft:
 percentage of microrafts for each grade (out of 529 microrafts per
 replicate) ##### ***Patterning Metrics\_Grading\_Means.csv*** Grade:
 micropattern grade [Centered (centered micropattern within microraft),
 Missing (missing micropattern), Border (micropattern contacting microraft
 border)] ArrayLoc: array location (middle, peripheral, total) Mean: mean
 percentage of microrafts for each grade (out of 529 microrafts per
 replicate) Stdev: standard deviation for percentage of microrafts for each
 grade #### **Plate vs Microraft Gastruloid Grading Count** Numbers of
 microrafts with fully developed gastruloids on multi-well plates and
 microraft arrays. ##### ***Plate vs Microraft Gastruloid Grading
 Count.csv*** Replicate: replicate number Substrate: substrate for
 gastruloid formation (plate or microraft) ArrayLoc: array location (middle
 or peripheral) Grade: gastruloid grade (good or malformed) Count: number
 of microrafts for each grade PercRaft: percentage of microrafts for each
 grade (out of 16 per array location per replicate) #### **Template
 Dimensions** Measurements of master template dimensions. ##### ***Template
 Dimensions\_Gap.csv*** Template: template number Gap: gap number Start:
 starting coordinate along x-axis of template cross-section in mm End:
 ending coordinate along x-axis of template cross-section in mm Gap Size:
 gap size calculated by subtracting “End” by “Start” in µm #####
 ***Template Dimensions\_Height.csv*** Template: template number Feature:
 feature number Height: height of feature in µm ##### ***Template
 Dimensions\_Width.csv*** Template: template number Feature: feature number
 Start: starting coordinate along x-axis of template cross-section in mm
 End: ending coordinate along x-axis of template cross-section in mm Width:
 Width of feature calculated by subtracting “End” by “Start” in µm #####
 ***Template Dimensions\_Means.csv*** Variable: Gap [gap width in µm],
 Height [feature height in µm], Width [feature width in µm] Template:
 template number or across all templates (all) Mean: replicate-level mean
 or mean across templates Stdev: replicate-level standard deviation or
 standard deviation across templates ### **File: EGOMRA Images.zip**
 Individual microraft (raft) images [see “Individual Raft Images”
 subdirectory] and stitched composite images [see “Stitched Images”
 subdirectory] of array for both fixed and living gastruloid analysis. For
 each replicate of the living gastruloids “Array 1” and “Array 2” refer to
 the euploid and aneuploid arrays, respectively. ###### *Fixed channels:*
 BF = brightfield (transmitted light) Blue = Hoechst 33342 Green = SOX2 Red
 = Brachyury (BRA) FarRed = CDX2 ###### *Living channels:* BF = brightfield
 (transmitted light) Blue = Hoechst 33342 Green = Calcein AM ##
 **Code/software** For code used, please refer to
 [https://github.com/ijan780/EGOMRA](https://github.com/ijan780/EGOMRA).