title: GSM3478484: Zf_07d_Control_B; Danio rerio; Bisulfite-Seq
library_construction_protocol: Total nucleic acids were purified using the Bio-On-Magnetic-Beads (BOMB) approach (Oberacker et al., 2018). Briefly, a guanidine isothiocyanate lysis buffer was used to homogenize cells, and then was combined with TE-diluted Sera-Mag Magnetic SpeedBeads (GE Healthcare, GEHE45152105050250) and isopropanol in volumetric ratio of 2:3:4 (beads:lysate:isopropanol). Beads were captured with a neodymium magnet and washed once with isopropanol, twice with 70% ethanol and resuspended in milliQ water. Bisulfite-converted genomic libraries were preparing using a modified post-bisulfite adapter tagging method. Briefly, bisulfite treatment was performed according to the EZ Methylation Direct Mag Prep kit (Zymo, D5044) instruction manual. To synthethise the first strand, we used converted DNA and 5'-biotinylated adaptor primers bearing a seven random nucleotides at its 3' end (BioP5N7, biotin- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNN). The first strand product was purified using streptavidin coated magnetic beads (ThermoFisher, 11205D) and alkaline denaturation.  Second strand DNA was synthesized using the immobilized first strand DNA and another adaptor primer also bearing seven random nucleotides at its 3' end (P7N7, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNN). Second strand DNA was eluted, amplified by 15 cycles of PCR and size selected by PEG-diluted SPRI beads. During PCR, unique molecular barcodes and sequences required for Illumina flow-cells binding were added to libraries using 1X HiFi HotStart Uracil+ Mix (KAPA, KK2801 and 10 μM indexed Truseq-type oligos). Library integrity was assessed on an Agilent 2100 Bioanalyzer and sequenced on a 250 bp single-end run on Illumina MiSeq. Post-bisulfite adaptor tagging (PBAT)
attributes.tag: GEO Accession
attributes.value: GSM3478484