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.

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.

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.

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. 

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

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).

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.