title: GSM3533032: WGBS_M_EB45.2; Daphnia pulex; Bisulfite-Seq
library_construction_protocol: Genomic DNA was extracted from the samples using MasterPure DNA purification kit (Epicentre, USA) following Athanasio et al. (2016). Libraries for sodium bisulfite treated samples were prepared following the EpiGnome Methyl-Seq kit (Epicentre, USA) protocol. Briefly, DNA samples (50 ng for sodium bisulfite converted and 20 ng for non-converted samples) were diluted in 9 μL of nuclease-free water, mixed with 2 μL of DNA synthesis primer and incubated at 95°C for 5 minutes. Samples were placed on ice and 5 μL of the mastermix (containing 4 μL of EpiGnome DNA synthesis premix, 0.5 μL of 100 mM DTT and 0.5 μL of EpiGnome polymerase) was added to each sample. The reactions were incubated as 25°C for 5 minutes, 42°C for 30 minutes and 37°C for 2 minutes. Then, 1 μL of exonuclease I was added to each sample and incubated for 10 minutes at 37°C, 3 minutes at 95°C followed by 2 minutes at 25°C. DNA samples were tagged by addition of TT master mix (7.5 μL of EpiGnome terminal tagging premix and 0.5 μL of DNA polymerase). Samples were incubated at 25°C for 30 minutes followed by 3 minutes at 95°C. Reactions were cooled to 4°C and purified using AMPure XP system (1.6x beads) (Beckman Coulter Inc., USA) as recommended. The final step of library construction comprised the amplification of the libraries and addition of barcodes. Each reaction contained 22.5 μL of the purified tagged DNA, 25 μL of FailSafe PCR premix E, 1 μL EpiGnome forward primer, 1 μL of EpiGnome index PCR primer and 0.5 μL of FailSafe PCR enzyme (1.25 U). PCR was performed according to the manufactures guidelines. Amplified samples were purified using AMPure XP beads (1x beads). Samples were re-suspended in 20 μL of nuclease-free water and quantity and quality of the sequencing libraries were assessed. Daphnia pulex libraries were sequenced using Illumina NextSeq-500 platform at the Centre for Genomics and Bioinformatics, Indiana University, USA.
attributes.tag: GEO Accession
attributes.value: GSM3533032