GSM4822590: ESC_TET_TKO_TET3_Ctr_42h_C; Mus musculus; Bisulfite-Seq
Total nucleic acids were purified using the Bio-On-Magnetic-Beads (BOMB) approach. 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. Purified DNA was subjected to bisulfite conversion using the EZ-96 DNA methylation Direct™ MagPrep kit (Zymo Research, D5044), according to the manufacturer's user guide, however, reagent volumes were scaled down to 25% of the recommended volume, with the exception of the final elution step which remained at 25 μL. To synthesise the first strand, we used converted DNA and 5′-biotinylated adaptor primers containing 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 synthesised using the immobilised first strand DNA and another adaptor primer also containing seven random nucleotides at its 3′ end (P7N7, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNN). Unique molecular barcodes and sequences essential for binding to Illumina flow-cells were added to the second strand DNA by PCR using 1 x HiFi HotStart Uracil + Mix (KAPA, KK2801) and 10 μM indexed Truseq-type oligos, amplified by 15 cycles of PCR and size selected by PEG-diluted SPRI beads. Library integrity was determined by agarose gel electrophoresis and sequenced on an Illumina HiSeq using single-end 100 bp chemistry.