Methods
DNA libraries for Illumina NGS were prepared with a two-step PCR approach. For this, the Nucleosome 1 - Linker - Nucleosome 2 sequence was split into two amplicons, one comprising the Nucleosome 1 - Linker and the second comprising Linker - Nucleosome 2. For library preparation, 1 μL of bisulfite-converted DNA was amplified in a first PCR reaction using barcoded primers and HotStartTaq DNA Polymerase (QIAGEN). In the second PCR reaction, 1 µL of PCR1 product was amplified using i5 and i7 indexing primers and Q5 polymerase (New England Biolabs). Successful amplification was verified by agarose gel electrophoresis. Samples were pooled in equimolar amounts, purified with NucleoSpin® Gel and PCR Clean-up kit and used for Illumina paired end 2×250 bp sequencing conducted at Novogene. Bioinformatic analysis of NGS data was conducted using a local instance of a Galaxy server. Obtained sequence reads were trimmed with the Trim Galore! Tool, discarding tails with a quality score below 20. Afterwards reads were paired using PEAR. Reads were filtered according to the expected DNA length using the Galaxy Filter FASTQ tool. The de-multiplexing was done by the selection of the reads with specific combinations of barcodes and Illumina adapters which were then then mapped against a corresponding reference sequence using bwameth (github.com/brentp/bwa-meth). Finally, the DNA methylation of individual CpG sites was computed using MethylDackel (github.com/dpryan79/MethylDackel). Here, the final analysed sequences are provided.
DNA sequencenes
Dinucleosome Linker-70
CGAGGTCGACGGTATCGATAAGCTTCTGGAGAATCCCCCAGCCGAGGCCGCTCAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGTTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCAGGTGTCAGATATATACATCCTGTAACGCTATCCGCGCCACGTCTACGCTNNNTACGAGAACGCCGAGACGTGCGAGCAGCGAAAGCGGCCGaCCTGGAGAATCCAGGTGCTGAGGCAGCTCAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGTTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACCACTCAGATATATACATCCTGTAAGGGCGAATTCCACATTG
Dinucleosome Linker-58(1)
CGAGGTCGACGGTATCGATAAGCTTCTGGAGAATCCCCCAGCCGAGGCCGCTCAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGTTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCAGGTGTCAGATATATACATCCTGTGCCACGTCTACGCTNNNTACGAGAACGCCGAGACGTGCGAGCAGCGAAAGCGGCCGaCCTGGAGAATCCAGGTGCTGAGGCAGCTCAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGTTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACCACTCAGATATATACATCCTGTAAGGGCGAATTCCACATTG
Dinucleosome Linker-58(2)
CGAGGTCGACGGTATCGATAAGCTTCTGGAGAATCCCCCAGCCGAGGCCGCTCAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGTTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCAGGTGTCAGATATATACATCCTGTAACGCTATCCGCGCCACGTCTACGCTNNNGAGACGTGCGAGCAGCGAAAGCGGCCGaCCTGGAGAATCCAGGTGCTGAGGCAGCTCAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGTTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACCACTCAGATATATACATCCTGTAAGGGCGAATTCCACATTG
CpG rich DNA
CTATGGAAACCCCTGTGGAGCTTCAGGGGCACGAGTGAGGCGGGCGCTGGCGGGCCAAGGTGACGAAGGCGCCTCCGGCTCTTGGGCCAGCGGACTGAGCGGTGGAGCAGAACTTGGGTGCCTCGGGGACCGCCAAAAAGTGGCCTTGTCCACTTCTCTGAG
Further information
The primers are provided in "NGS_Primers"
A compilation of the sequencing reads provided here is given in "NGS_Table"
References
The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. Nucleic acids research 2022, 50, W345-W351, doi: 10.1093/nar/gkac247
Albrecht, C., Bashtrykov, P., and Jeltsch, A. (2024) Amplicon-Based Bisulfite Conversion-NGS DNA Methylation Analysis Protocol. Methods Mol Biol 2842, 405-418, doi: 10.1007/978-1-0716-4051-7_21
For all details regarding furhter analysis and interpretation of the data, refer to the corresponding manuscript (Gutekunst et al., 2026), which is connected with this data entry.
