README

DEVELOPMENTAL PROGRESSION

- This project produced imaging data optained on an Olympus confocal microscope 
- Pescoids were exposed to different conditions and imaged
  - Usually, in BF and with GFP signal (mezzo, H2B, gsc, ...) (2 channels)
  - Z-slices of around 5 um (~ 15 z-slices) to cover tissue before light diffraction decreased resolution
  - Time-points of around 30 time-points, starting at around 6 hpf with 30 min intervals until 24 hpf depending on sample
- Imaging of pescoids in time-lapse was performed with 10x magnification and 512x512 resolution
  - Larger files exceeding 1GB killed the imaging session (microscope is old)

- Imaging data was obtained in OIR format
> ImageJ_OIR_to_TIF.ijm
  - ImageJ Macro
  - Transforms OIR files to TIF files that are easier to work with
- One TIF file = one pescoid sample

- Have a quick look at the data
> ImageJ_quick_check_pescoids.ijm
  - Edits images
  - Creates AVI file to have a look at time-lapse data
  - Creates JPG files at 6 hpf and 12 hpf to have a quick look at the data

- Experimental conditions: chemical perturbations or mechanical perturbations by 3D confinement 
> ImageJ_segment_pescoids.ijm
  - Analysis of developmental dynamics in different conditions:
    - Elongation: Aspect ratio, major axis length
    - Fluorescence fraction: GFP-positive area normalized to overall BF area of the pescoid
      Normalization is necessary since different conditions induce different surface areas
      For example, strongly confined pescoids are flattened and increase in area
  - Segmentation of the BF area (entire pescoid)
  - Segmentation of GFP-positive area
  - Generates one folder for one TIF file (1 TIF file = 1 pescoid / sample)
    - Each folder contains 1 JPG file per time-point
    - AVI file with time-lapse data as segmentation
    - CSV file with all results and measurements called "Results.csv"
- "Results.csv" from last sample contains all measurements of all samples in that folder / condition
- "Results.csv" needs to be copied in general folder for analysis

- Segmented samples have to be analyzed and plotted
- "Results.csv" needs to be loaded for each condition (control, condition1, condition2, ...)
> R_analyze_segmented_pescoids.Rmd
  - Load necessary libraries
  - Adjust parameters in first lines
  - Add conditions if necessary
  - Code contains many explanations, check for details
  - Result: saves plots as PDFs in folder
    - AR over time
    - AR over major axis length
    - Fluorescence fraction over time 

TISSUE FLOW
  (with great support from Sham Tlili and Vanessa Weicheslberger)

- Some samples were analyzed for tissue flow
- Here, nuclear markers were used with lower time-intervals (2-10 min)
- Fewer z-slices in the periphery or center of the pescoid
- Higher magnification (20x) and resolution with fewer samples to not exeed 1GB limit of microscope
- Follow the instructions in the manual. This explains the steps to pre-process the data in ImageJ and how to run the MatLab scrip.

> MatLab_tissue_flow_manual.pdf

- Once the pre-processing is complete and the masks are generated, the MatLab script can be run
- The scrip should be run for each individual pescoid to set the parameters accordingly
> MatLab_tissue_flow.m

- After running the script for each pescoid, run the measurement of the average velocity
> MatLab_tissue_flow_average_velocity
- This gives values that you can plot over time by using this R script
> R_tissue_flow_velocity_over_time.Rmd

SCI RNA SEQ
  (with great support by Michael Dorrity)

- bulkRNAseq data was generated using CRG gene core
- Projection of our data in reference data set by White 2017
> R_data_projection_in_reference.Rmd
- PCA was generated by Patryk Polinksi

- sciRNAseq data was annotated by Nick Marschlich with great support of Michael Dorrity
- Analysis based on Monocle3 package in R
- First step was to annotate 39 initial clusters
> R_PAc_annotation_specificity.Rmd
- Then the data was analyzed for developmental progression and subclusters
> R_PAc_clsuters.Rmd
- Next steps will focus on differential gene expression