Open Access additional raw data


Single-Cell RNA-Seq Reveals Transcriptional Heterogeneity 
in Latent and Reactivated HIV-infected Cells
Monica Golumbeanu1,2, Sara Cristinelli3, Sylvie Rato3, Miguel Munoz3, Matthias Cavassini4, Niko Beerenwinkel1,2, *, Angela Ciuffi3, *
Doi : 10.1016/j.celrep.2018.03.102

Experimental procedure:

Quantitative analysis of GFP expression in TCR-treated cells
Cells were prepared for single cell analysis at the Genome Technology Facility (GTF) of the University of Lausanne. Cells were loaded on Fluidigm C1 IFC plates (5-10 um), with run ID smart33, smart34 and smart35, corresponding to untreated, SAHA- and TCR-treated conditions respectively. After single cell capture on the Fluidigm C1 IFC plate, each chamber was inspected visually by microscopy and pictures were captured with a Zeiss Axiovert 200 M fluorescence microscope equipped with a Roper Scientific CoolSnap HQ camera using a Plan-Neofluar 10X lens (smart34 run) or 20X lens (for smart35 run). For each capture chamber, pictures in bright field and FITC channel were taken with the MetaMorph 6.3 software. Picture analysis was then performed using ImageJ 1.50b software (open access software: website). Brightness and contrast were adjusted for qualitative assessment of the pictures. The Corrected Total Cell Fluorescence was calculated as follows: Briefly, for every raw picture, the capture region containing a single cell was manually outlined using the rectangle selection tool. The integrated density and the area of the region were measured with the ImageJ measure tool. The same outlining procedure was applied to a background zone close to the capture region and the mean fluorescence was measured with the ImageJ measure tool. The Corrected Total Cell Fluorescence (CTCF) was then calculated using the following equation:
CTCF = Integrated Density (of the selected cell) – (Area (of the selected cell) * Mean Fluorescence (of the background))
Three replicate measurements were performed for each cell capture site, using three different background areas surrounding the cell capture site.





Attached files
* Fluorescence_quantification_TCR_SAHA_raw_data.xlsx
* Microscopic pictures (TIFF format) -> to open with ImageJ or Fiji
o X_34_SAHA-treated_ pictures
o X_35_TCR-treated_ pictures

The Fluorescence_quantification_TCR_SAHA_raw_data.xlsx excel file contains details of the measurements taken for each picture.
It is structured in two sheets named: 
* SMART_34_SAHA_Raw measurement: containing fluorescence assessments of SAHA-treated cells using ImageJ software.
* SMART_35_TCR_ Raw measurement: containing fluorescence assessments of TCR-treated cells using ImageJ software.

Each sheet presents a table, which is divided in 12 columns:
1. Cell ID: specifying the run_numberID (smart xx) and the sample number ID (Cxx). Example: Smart35_C05 should be read as run number 35, cell sample number 5.
2. Number of Estimated Cells/Capture-site: number of cells visually assessed after cell separation on fluidigm plate in each capture site. This information is subjective and is only indicative of the cell presence, but is not taken in consideration for further analyses.
3. Cell area vs Background (Bg): indicates if the measurement is taken on the capture site containing a single cell or on the background. The analysis is performed three times (a,b,c), using different random locations for the background, and slightly different locations on the capture site. 
4. Size of selected Area: area size of rectangle selection tool used to measure fluorescence intensity.
5. Mean Grey Value : average grey value within the selected area. This is the sum of the grey values of all the pixels in the selection divided by the number of pixels. Reported in calibrated units.
6. Min Grey Value: minimal grey value within the selected area.	
7. Max Grey Value: maximal grey value within the selected area.	
8. IntDen (Integrated Density):	 relative assessment of the fluorescence intensity. It is calculated using the following formula:
      IntDen = Size of the selected area * Mean Gray Value
9. CTCF (Corrected Total Cell Fluorescence): Normalized assessment of the fluorescence intensity. It is calculated according to the following formula: 
CTCF = Integrated Density (of the selected cell) – (Area (of the selected cell) * Mean Fluorescence (of the background))
10. CTCF mean: mean of the three CTCF values obtained from the three replicate measurements.
11. CTCF STDEV: standard deviation of the three CTCF values obtained from the three replicate measurements.
12. ID of Corresponding File: name of the image file corresponding to the analysis.



Microscopic Pictures		
Individual microscopic pictures are named X_34_SAHA_treated_pictures and X_35_TCR  treated_pictures for the SAHA-treated and the TCR-treated condition respectively.
Each file name is followed by the microscopic channel filter, i.e. either BRIGHT or GFP, to indicate that the picture was taken in the brightfield or in the FITC channel (green).
Each file is thus named according to the following pattern:
      runID(smartxx)_sample number(sxx)_ magnification_ treatment_filter(BRIGHT/GFP)
Example
      smart34_s4_ 10X_ SAHA _GFP
This should be understood as picture taken in the run 34 at cell capture-site number 4, at 10X magnification. Samples have been treated with SAHA and this is the picture in the FITC channel.