title: GSM2562938: Female4_Liver; Callorhinchus milii; Bisulfite-Seq
library_construction_protocol: Elephant shark tissue samples were provided by commercial fishermen from recently deceased animals. DNA was purified using a modified magnetic bead approach (Deangelis et al., 1995). Briefly, cells were first homogenised in “GITC” lysis buffer (4 M Guanidine thiocyanate, Sigma G6639; 50 mM Tris, Thermo 15568-025; 20 mM EDTA; Thermo 15575-020; 2% Sarkosyl, Sigma L9150-50G; 0.1% Antifoam, Sigma A8311-50ML) and this lysate mixture was then combined with TE-diluted Sera-Mag Magnetic SpeedBeads (GE Healthcare, GEHE45152105050250) and isopropanol in a volumetric ratio of 2:3:4 respectively. Following capture with a neodymium magnet, beads were washed once with isopropanol, twice with 70% ethanol and resuspended in filter-sterile milliQ H2O. WGBS-seq was undertaken using a post-bisulfite adapter tagging (PBAT) method adapted from Peat et al., 2014. Briefly, 50-100 ng of purified DNA was subjected to bisulfite conversion using the Imprint DNA modification kit (Sigma, MOD50).  Converted DNA underwent first strand synthesis with a biotin labelled adapter sequence possessing seven random nucleotides at its 3' end (BioP5N7, biotin- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNN). The product of first strand synthesis was captured using streptavidin-coated Dynabeads (Thermo, 11205D) and magnetic immobilisation. Double stranded DNA was created using the immobilized first-strand as a template and an additional adapter also possessing seven random nucleotides at its 3' end (P7N7, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNN). Unique molecular barcodes and sequences necessary for binding to Illumina flow-cells were added to libraries by PCR using 1X HiFi HotStart Uracil+ Mix (KAPA, KK2801 and 10 μM indexed Truseq-type oligos, with thermal cycling as follows: 12*(94°C, 80 sec; 65°C, 30 sec; 72°C, 30 sec).
attributes.tag: GEO Accession
attributes.value: GSM2562938