Full scRNAseq analysis
Data the SCC collected on both inDrops and 10x instruments is available here:
For full service bioinformatic analysis service we suggest you work with the Chan Bioinformatics Core.
Number of cells: 25,000 – 30,000 at a minimum.
Cells should be at a density of more than 100,000 cells/ml in at least 150ul.
Viability: Ideally greater than 90%
Buffer conditions: Buffer should NOT contain Mg2+, Ca2+ or EDTA.
Most of users resuspend their cells in PBS (without Mg2+, Ca2+ or EDTA) + BSA (upto 1%)
- Single-nucleus RNA sequencing can be run with inDrops. Several users perform this routinely.
- A standard inDrops day encapsulates 6,000 cells per sample for up to 6 samples.
- Optiprep information
- CITE-seq / Cell Hashing have succesfully run on both inDrops and 10x.
10x Single-cell sample prep resources
10x Genomics provides a tremendous amount of sample prep protocol advice. Most of the sample prep requirements for 10x are the same for inDrops. This is a great resource to use for sample prep optimization.
There are a variety of scRNA-Seq and scNuc-Seq methods available.
Click here for a search of "single cell dissociation scRNA-Seq"
InDrops Method Publications
Zilionis R, Nainys J, Veres A, Savova V, Zemmour D, Klein AM, and Mazutis L. 1/2017. “Single-cell barcoding and sequencing using droplet microfluidics.” Nat Protoc, 12, 1, Pp. 44-73.
- Klein AM, Mazutis L., Akartuna I, Tallapragada N, Veres A, Li V, Peshkin L, D. A. Weitz, and MW Kirschner. 2015. “Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.” Cell, 161, Pp. 1187-1201.
Most users sequence their libraries on a NextSeq 75 bp run:
Read 1: 61
Read 2: 14
Index 1: 8
Index 2: 8
It is common to have 70-80% valid reads after filtering for valid cell barcodes.
Sequencing on a their libraries on a NovaSeq 100 bp run:
Read 1: 86
Read 2: 14
Index 1: 8
Index 2: 8
Attention!!! illumina has come out with new NovaSeq kits (V1.5). We have seen that inDrop libraries are not sequencing correctly for Index read 2 on these NovaSeq V1.5 kits. V1 NovaSeq kits will sequencing inDrop libraries with no issue. Solution! V1.5 kits will sequence inDrop libraries properly if you use a custom recipe for the NovaSeq settings that sequences the libraries using the grafted primer on the flow cell. BPF, Bauer Core, and DFCI cores are aware of this, but you should remind them if sequencing inDrop libraries that they need to use a custom recipe or V1 kit. If sequencing with any other core please have them contact us for details.
After several runs of inDrops libraries on the NovaSeq, we have identified that there are sometimes issues with NovaSeq data from using the standard adapter trimming options. The samples are sequencing fine, but inDrop runs on the NovaSeq require that you recieve UN-TRIMMED Fastq files. Adapter trimming seems to be causing some loss of valid reads causing you go only get 6 bp of some of the 8 bp index read as well as issues on Read 1. We have seen that Read 1 sometimes need adapter trimming to map well, but this needs to be done after Fastq generation. Please let us know if you experience any issues on a NovaSeq run so we can help troubleshoot and understand the issues for all inDrops users.
- Full day or half day services are available. To qualify as a half day run, you must have 3 or fewer samples and be collecting no more than 15,000 cells in total.
- Run preparation begins 1 hour in advance of the scheduled time. To avoid reagent and service fees, please notify us of any delays or cancellations at least 2 hours in advance of your start time.
- If you are more than an hour late for your scheduled run, the SCC staff reserves the right to cancel your run and charge you for the reserved time and reagents used in preparation for your run.
- The SCC will hold on to backup encapsulated cells for 1 year after your run date. If you need your backup emulsion please retrieve it from the SCC within a few months of your run.