FindingNemo Extraction 4: Circulomics Kit
Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose
ultra-long sequencing
cohex
glass bead
nanopore
MinION
UHMW DNA
Monarch
Circulomics
phenol
SDS
CTAB
GM12878
Whatman
PromethION
Nanobind
Abstract
This is a sub-protocol designed to extract/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using the Circulomics Nanobind CBB Big DNA Kit .
A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters.
The Circulomics Nanobind CBB Big DNA Kit was initially the recommended extraction protocol for UL sequencing using SQK-ULK001.
We tested this sub-protocol in human cell line , with standard input cells of 6 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.
Before start
Things to observe at all times:
- Excessive and vigorous pipetting and vortexing should be avoided as these may shear the DNA.
- Make up buffers with nuclease-free water to avoid introducing nucleases to solutions.
- Avoid unnecessary heating and freezing; isolated DNA should be stable for storage in the fridge for months.
Steps
UHMW DNA Extraction
This protocol is using the Nanobind CBB Big DNA Kit (see Materials).
Follow the manufacturer’s instructions as described here.
After overnight incubation at room temperature, quantify the DNA as per "UHMW DNA QC" and check homogeneity by calculating %CV values.
Store at 4oC or continue to UL Library Preparation as per Section " Modified ULK001 ".
If only SQK-RAD004 is available, follow library preparation as in Section " Modified RAD004 " or "KrazyStarFish (KSF) ".
4°C
