This could be caused by low cell numbers,RNA degradation in the tissue or many other factors induced during the experimental procedures. Also, it may be caused by sample loss during experimental procedures or insufficient cDNA release during cDNA release step.

The center-to-center distance between two spots is 500 nm.

Main peak of cDNA amplification products for tissues with RIN value > 7 locates at around 1000-1500 bp. The fragments of final library range from 200-600 bp.

Store it in TE buffer at 4℃.

Library structure is indicated in the figure below:

Use the following configuration to perform paired-ended sequencing run:
Read 1: 50 cycles (25 cycles for barcode + 15 dark cycles for fixed sequence +10 cycles for MID),
Read 2: 100 cycles
Sample barcode:10 cycles
The library construct is compatible with our DNBSEQ platform. Users are free to try out other platforms.

After ssDNA/DAPI staining, the fluorescent signal contrast between the capturing probe loaded area and unloaded area allows visualization of the track lines.

Incorrect. Long permeabilization time might cause RNA diffusion. After complete tissue removal, under the same exposure setting, the optimal permeabilization time should be identified from observations of complete tissue morphology, strong fluorescent signal, and minimum signal diffusion.

STOmics R&D team has compared and tested various commercialized staining reagents and found that ssDNA staining has the least effects on the downstream mRNA capture rate. In addition, ssDNA staining allows visualization of track lines on the

STOmics Permeabilization Set is used for optimizing permeabilization conditions for a specific tissue of interest. All images should be taken under the same exposure time, brightness, and contrast settings. In order to judge the permeabilization performance with different permeabilization times more easily, it is recommended to use ImageJ software to process the images first.
Method 1: Drag all the images with different permeabilization times into ImageJ, go to Image -> Stacks -> Stack to Images (F2 could also be used to generate stacks). Click OK to generate a stack. Go to Image->Adjust -> Brightness /Contrast. Press Auto to adjust image parameters of the stack uniformly. Use the arrow key left/right to go through the slices in a stack. If you are not satisfied with the auto adjustment, click Reset, and adjust the image parameters manually until you select the image parameters with obvious contrast. Choose the one that generates the clearest tissue morphology and strongest fluorescent signal as the optimal permeabilization time, and the obtained permeabilization time can be a time range, such as 9-12 min.
Method 2: Open 'ImageJ' and open the tif images of the same tissue in the order of shortest to longest permeabilization time points. Click on the 'Image' toolbar, select 'Adjust', choose 'Brightness/ Contrast', adjust 'Maxiumu' to make the image brightness clear. Create a rectangular selection of the whole tissue area, click 'Analyze' toolbar, select 'Measure', and get the fluorescence grayscale of the tissue area. When measuring another picture, keep the images to be measured as the current selected state, select 'Ctrl+shift+E' to paste the measurement box, drag the measurement box to the new tissue area and make slight adjustment. Repeat previous steps to get the fluorescence grayscale of the tissue area. If total RNA was used as positive control, use a circular selection box to circle out the fluorescent area. Each dot should be measured individually and calculate the average gray value. The measured grayscale values are then used for the determination of the optimal permeabilization time point.

The QubitTM ssDNA Assay Kit is designed to quantify ssDNA. However, it also detects dsDNA and RNA. For more information, kindly refer to the manufacturer's user guide.https://www.thermofisher.com/order/catalog/product/Q10212.