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The DNA extracted from the CD4⁺ naïve cells was prepped for mRRBS on an Illumina HiSeq3000 according to the Boyle et al. mRRBS protocol (20 (link)) using the Diagenode Premium RRBS kit (Diagenode, Seraing, Belgium) (21 (link)). The mRRBS libraries (N = 48) were sequenced with indexes from the Diagenode Premium RRBS kit as 50 base pair, single end reads with 20% PhiX spike in and 6 samples per lane.
The CD4⁺ memory cells were prepped for mRRBS on Illumina HiSeq2500 at the Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, using an in-house protocol based on Boyle et al. (20 (link)) and Gu et al. (22 (link)). The libraries (N = 48) were sequenced as 50 base pair single end reads with 10% PhiX spike in and 6 samples per lane. Achieved Phred quality score was >28 in all retained reads from the mRRBS sequencing.
One sample (technical replicate) was sequenced on both platforms (HiSeq 2500 and HiSeq 3000) and was compared to determine how the different sequencing platforms would impact the results. The correlation was 0.9341 for this sample, and hence we found the results to be comparable (Table 2).
The generated methylation data has been deposited to Gene Expression Omnibus (accession number GSE135770).

The DNA extracted from the CD4⁺ naïve cells was prepped for multiplexed reduced representation bisulfite sequencing (mRRBS) on Illumina HiSeq3000 according to the Boyle et al. mRRBS protocol (24 (link)), using the Diagenode Premium RRBS kit (Diagenode, Seraing, Belgium) (25 (link)). The mRRBS libraries (N=18) were sequenced with indexes from the Diagenode Premium RRBS kit as 50 base pair, single end reads with 20% PhiX spike control and 6 samples per lane.
The CD4⁺ memory cells were prepped for mRRBS on Illumina HiSeq2500 at Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, using an in-house protocol based on Boyle et al. (24 (link)) and Gu et al. (26 (link)). The 18 libraries were sequenced as 50 base pair single end reads with 10% PhiX spike control and 6 samples per lane.
Achieved Phred quality score was >28 in all reads used from mRRBS sequencing, for both cell populations.
One sample (technical replicate) was sequenced on both platforms (HiSeq 2500 and HiSeq 3000) and was compared to determine how the different sequencing platforms would impact the results. The correlation was 0.9341 for this sample, and hence we found the results to be comparable.

Reduced representation bisulfite sequencing (RRBS) was used to analyze the DNA methylome, which is preferred over whole genome bisulfite sequencing due to the enrichment of areas with high CpG content [58 (link)]. The RRBS libraries were prepared using the Diagenode Premium RRBS Kit (#C02030032, Diagenode, Denville, NJ) with the following exceptions: 200 ng of genomic DNA was used for input and the equation cycle threshold (Ct) + 2 was used to determine the number of PCR cycles needed for final library amplification. AMPure XP Beads (Beckman Coulter, Brea, CA) were used for all steps in the Diagenode protocol requiring size selection or clean-up of products. Final libraries were stored at -80° C until sequencing. Libraries were prepared in two separate batches, 2-, 6-, 12-months and 4-, 5-, 9-, 14-months, respectively.