MEP-LINCS HMEC240L_SS4 Analysis

date: 2017-08-18

Introduction

The LINCS HMEC240L_SS4 experiment was performed with HMEC240L cells grown in 8 8-well plates. The analyzed endpoints include DAPI, KRT5, KRT19 and EdU. Color images of the cells at each spot were gathered on a Nikon automated microscope.

Intensity, position and morphology data are gathered for each cell, merged with the experiment metadata, normalized with RUVLoessResiduals, filtered and summarized.

Spot Cell Count Analysis

The spot cell count analysis identifies MEPs with extreme population sizes. The normalized spot cell counts in the plot below are summarized by the median and standard error of their replicates. Hovering over the the interactive plot below shows the MEP identities. Clicking and dragging over a section of the plot will zoom into the selected location. Double clicking on the zooomed plot will restore the original plot.

Normalized Spot Cell Counts

The interactive heatmaps below are arranged by unsupervised clustering of the rows and columns and colored by the normalized spot cell count. Clicking and dragging across any subsection will zoom in on that section. Double clicking on the zoomed image will return to the full heatmap.

DNA Content Analysis

All cells are stained with DAPI and autogated as DNA 2N or 4N. The proportion of 2N and 4N cells at each spot is calculated and will always sum to 1. The proportions are logit transformed then RUV and loess normalized then back transfomred to a proportion. Lower normalized values have smaller 2N populations and therefore larger 4N populations.

Normalized DNA 2N Heatmaps

The interactive heatmaps below are arranged by unsupervised clustering of the rows and columns and colored by the normalized DNA 2N proportions. Clicking and dragging across any subsection will zoom in on that section. Double clicking on the zoomed image will return to the full heatmap.

Proliferation Analysis

The proliferation analysis method is as follows:
First, the cells within each well are auto-gated into Edu+
The proportion of EdU+ cells at each spot is calculated
The proportions are logit transformed then RUV and loess normalized
The normalized proportions are then summarized by the medians of their replicates.

Normalized EdU-based Proliferation Heatmaps

The interactive heatmaps below are arranged by unsupervised clustering of the rows and columns and colored by the normalized EdU+ proportions. Clicking and dragging across any subsection will zoom in on that section. Double clicking on the zoomed image will return to the full heatmap. The first heatmap is for the full dataset and the second is for the HF dataset.

Lineage Marker Analysis

This staining set includes the lineage markers KRT5 or KRT14 which are associated with basal cells and KRT19 which is associated with luminal cells. This analysis evaluates the lineage responses through a ratio of the median intensity of luminal to basal cell markers.

Principal Components Analysis

A PCA dimension reduction is performed on the 73 normalized features that have non-zero variance. Hovering over the datapoints will show the MEP values.

tSNE Analysis

A tSNE dimension reduction is performed on the 73 normalized features that have non-zero variance. Hovering over the datapoints will show the MEP values.