Q
Why does a second sharp peak appear after the main peak in the ADT library fragment analysis?
A
The second sharp peak that appears after the main fragment peak is a product of non-specific barcode amplification. The non-specific amplicons do not affect the sequencing output or subsequent analysis.
Q
In the ADT product fragment distribution analysis, a broad peak between 300-500 bp is observed. What does this indicate, and will it affect subsequent analysis?
A
The broad peak between 300-500bp indicates the presence of bubble products, which are hybrid double-stranded molecules formed during ADT library preparation from partially homologous fragments with complementary adapter sequences. These bubble structures do not affect sequencing or data analysis, but they can influence library quantification. After sample barcode is added through the PCR step, most bubble products are reduced. If high levels are still observed after PCR, 2.0X purification can be done, followed by an additional two cycles of amplification to further decrease bubble products.
Q
Why the PR enzyme concentration is changed from 1X to 0.5X in Stereo-CITE V1.1?
A
In certain tissues, lowering the working concentration of the PR enzyme from 1X to 0.5X enhances the stability of protein capture and minimizes diffusion.
Q
Why was the tissue fixation time changed from 10 minutes to 5 minutes when updating from Stereo-CITE V1.0 to V1.1? Does the incubation time require strict control?
A
Extending the tissue fixation time is more likely to cause mRNA cross-linking. In the Stereo-CITE V1.1 workflow, the de-cross-linking step has been removed to avoid affecting the overall mRNA capture; thus, the tissue fixation time has been set to 5 minutes, and the incubation time should be precisely controlled.
Q
How many isotype control antibodies should be used?
A
The number of isotype control antibodies is not determined by the total number of target antibodies but rather by the host species of the primary antibodies and the classification of their heavy and light chains. For instance, if 20 antibodies are used in a mouse sample, all sourced from Rat, with heavy chains of IgG2a, IgG2b, and IgG1, and light chains of κ, three isotype control antibodies should be included: Rat IgG2a κ, Rat IgG2b κ, and Rat IgG1 κ,.
For TotalSeq™-A series antibodies, the selection of isotype controls can be referenced at the following link: https://www.biolegend.com/en-us/search-results?PageSize=25&Category=ISO_CTRL&Format=TOTALSEQ_A
Q
Can pre-mixed commercially available antibody cocktails be used for this experiment?
A
Yes, currently validated mouse antibody cocktails include the TotalSeq™-A Mouse Universal Cocktail, V1.0 (Biolegend, Cat. No. 199901, for mouse samples, consisting of 119 antibodies) and the TotalSeq™-A Human Universal Cocktail, V1.0 (Biolegend, Cat. No. 399907, for human samples, consisting of 154 antibodies).
Q
Do Biolegend antibodies with a Poly A tag compete against target mRNA molecules on Stereo-seq chip? As the target protein number increases, does this enhance the competition for capture probes between the PolyA on antibodies and the mRNA from the tissue?
A
In theory, competition exists. However, there are sufficient probes on each DNB spot to effectively mitigate this competition. It has been confirmed that there is no impact on mRNA capture in a comparison of two adjacent tissue sections: one section with no antibodies applied, and the other with a high-plex antibody panel (for example, the 100+ plex Biolegend antibody panel) applied.
Q
How to distinguish the background signals from the signal in a protein expression heatmap?
A
It should be determined based on existing knowledge of each protein expression pattern in cells within the relevant field.
Q
How can we determine the spatial location information of individual proteins in a single-color immunofluorescence image containing multiple proteins?
A
The single-color immunofluorescence image obtained from the fluorescence microscope shows the combined signals of multiple proteins, making it unsuitable for determining the spatial location of individual proteins.
Q
Concerning the secondary antibody incubation and imaging process, can fluorescent imaging of secondary antibody staining be omitted without impacting the experimental results? If imaging can be omitted, is it also possible to skip the secondary antibody incubation?
A
The fluorescent imaging of secondary antibody staining is an important QC step in the biochemistry workflow. We strongly recommend that customers perform this step to ensure that primary antibodies successfully bind to the target antigens on tissues. If users choose not to take images, they can skip the secondary antibody incubation and imaging steps. However, STOmics will not provide after-sales service or compensation for any experimental result abnormalities caused by skipping this step.
