Ever fancy a go with a new technique only to find that the methods section of the paper is holding out on the most intimate details? While single-cell bisulfite sequencing (scBs-seq) has been lusted after by many a researcher, mingling with those single cells has proved a bit more challenging. But fear not, the labs of Wolf Reik & Gavin Kelsey at the Babraham Institute (UK) have put out a much-needed protocol to help you get your single-cell bisulfite sequencing (scBs-seq) game on.
Previously, the group flirted with our scientific hearts by showing that scBS-seq captures up to 50% of CpGs in the mouse genome. Now, they are offering to play wingman for other would-be sequencers by describing a step-by-step playbook with all their latest optimizations, which can be done in under a week with the right mix of molecular biology and bioinformatic moves.
Their scBS-seq technique makes use of a modified post-bisulfite adaptor tagging (PBAT) method, which involves additional amplification steps. A post-bisulfite preamplification minimizes the loss of informative DNA fragments critical for your library complexity, while a final library amplification makes sure you have enough material to feed that hungry Illumina sequencer.
The library preparation step ranges from 2-3 days, while sequencing and analysis takes anywhere from 2-11 days. This protocol comes with a detailed list of reagents and equipment, step-by-step instructions for all the wet lab work, a list of needed software, and bioinformatic commands. It also comes with details about quality control and trouble-shooting advice for those tricky steps.
The protocol covers:
- Cell lysis and bisulfite conversion
- Preamplification and adaptor tagging
- Library amplification
- Methylation calling
While the playbook doesn’t cover all possible downstream bioinformatic analysis, it goes over the essential data processing needed to wet your dry lab appetite, which includes:
- Sequencing quality control
- Single-end, nondirectional alignment needed for PBAT
- Removing duplicate reads
- Extracting the methylation calls
The protocol also comes with options to keep your single-cell relationship open. Options include the use of an automated liquid handling robot, the Agilent Bravo platform, which allows for time-efficient, parallel processing of up to 96 samples at a time. It also offers an optional protocol for their recent parallel single-cell bisulfite and RNA-seq method. They also reveal the modifications needed to adapt the method from single cells to low-input samples ranging from tens to thousands of cells.
In addition to the protocol itself, the paper provides technical background, experimental design considerations, a comparison of the currently available single-cell bisulfite sequencing methods, and a wish list that includes the ability to discriminate 5hmC.
Go learn how to mingle with your single cells over at Nature Protocols, February 2017