This section contains information about ISSAAC-seq using the droplet workflow. Here, the 10x Genomics Single Cell ATAC kit was used, but any droplet systems with a Nextera capture sequence should work, such as Bio-Rad and HyDrop.
| Read | Description | |
|---|---|---|
| ATAC | Read 1 (R1) | gDNA read |
| Read 2 (R2) | gDNA read | |
| Index 1 (I1) | Sample index (not needed for analysis) | |
| Index 2 (I2) | i5 index (cell barcodes) | |
| ---------- | -------------- | -------------------------------------------------------------------------------------------- |
| RNA | Read 1 (R1) | cDNA sequence, might have some adaptor contamination depending on how long you sequence it |
| Read 2 (R2) | The first 10 bp are UMIs, the rest are ignored as they are mostly dT | |
| Index 1 (I1) | Sample index (not needed for analysis) | |
| Index 2 (I2) | i5 index (cell barcodes) |
Check this page to see how a step-by-step guide of how the libraries are generated. In this workflow, single nuclei are caputred using the droplet microfluidics after in situ treatment. Since we use the 10x Genomics Single Cell ATAC kit for both RNA and ATAC libraries, the sequencing configuration is the same for both modalities:
> 50 cycles for Read 1 (R1)
> 50 cycles for Read 2 (R2)
8-10 cycles for I1 (i7) <-- This is the sample index
16 cycles for I2 (i5) <-- This is the cell barcode
That means you sequence the libraries as if they are 10x scATAC-seq libraries. Only R1, R2 and I2 are needed for the analysis. You can basically process them using the Snakefile provided in this repository.