Individually hashed samples were counted using a hemocytometer and pooled in equal ratio at high concentration. Pooled sample was strained through a 70 µm cell strainer and counted again using a hemocytometer. To achieve approximately 20,000 cells after doublet removal, cell concentration was adjusted to 1314 cells/µL to achieve the target of 41,645 cells in 31.7 µL for super-loading of the 10X Chromium Chip A. Gene expression (using 5′ v1 chemistry; 10X Genomics) and ADT and hashtag-oligo (HTO) libraries were constructed using reagents, primers, and protocol from the published ECCITE-seq protocol (Mimitou et al., 2019). All libraries from the titration run were sequenced together with other samples on an Illumina NovaSeq6000 S1 flow cell. The post-titration (adjusted) sample (using 5′ v1.1 chemistry; 10X Genomics) was multiplexed and sequenced together with other samples not included in this study on Illumina NovaSeq6000 SP and S1 flow cells.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

Salina Dominguez
 
Would like to request the super-loading of 10x Chromium protocol used in study
2021-11-17 12:39:29 Reply
Terkild B Buus
 Author
Thank you for your interest in our manuscript. The super-loading of 10X chromium concept is published in the original hashing manuscript (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1603-1) as well as the original ECCITE-seq manuscript (https://www.nature.com/articles/s41592-019-0392-0). In short, it is simply loading more cells than recommended by the manufacturer. This increases the amount of doublets, but due to sample multiplexing in the hashing and ECCITE-seq protocols, many of these doublets can be identified and removed in silico (but are still sequenced).
2021-11-29 05:15:26 Reply



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.