Personally I have good experiences working with ssDNA, and would therefore choose a library prep kit adapted to this. For RNA Seq we used the the Ion Xpress Plus Fragment Library Kit (Rev. A) of Life Technologies. For ssDNA with MeDIP-Seq we used the Accel-NGS® 1S Plus DNA library Kit for Illumina® Platforms (Swift Biosciences). Different platforms, different kits, but good results in both cases. Additionally Swift Biosciences was very responsive and helpful when troubleshooting. 

Hi Maxime,

I don't have experience with Accel-NGS, but had a quick look on the workflow and it appears that that kit is suitable if you have degraded DNA; but Fleur must have more experience with that kit. my concern is that if you sscDNA of good quality (containing long frangments) processing directly with the kit would generate long fragment that you won't be able to sequence. in that case you may need to sonicate your cDNA before proceeding to library construction. My advice is to first run your cDNA on a bioanalyzer to check the fragment's size of your samples and then adjust your protocol accordingly.

the advantage of synthesizing dscDNA it that you can generate strand specific libraries, which will give an indication on the sens of transcription (useful to look at anti-sens transcripts for instance). here are the ingredients that I'm using:

1. Prepare the 2nd strand reaction on ice as follow:

·         RNA/cDNA hybrid 15 ul

·         10x NEB buffer 2  2 μL

·         dUTP mix (10mM dA, dC, dG and 20 mM dU) 1 μL

·         RNase H 0.5 μL

·         DNA polymerase I 1 μL

·         DTT (100mM) 0.5 μL

Final volume: 20uL

2. Incubate at 16°C for 2.5 hours

then proceed to library preparation: end-repear, dA-tailing and adapter ligation. before PCR amplification (to insert the illumina index) you treat your samples with USER to excise the fragment containing Us.

however, as mentioned above you may need to sonicate your cDNA before proceeding to these steps.

Good luck