title: GSM6205171: IFCa5; Mus musculus; Bisulfite-Seq
library_construction_protocol: Blastocysts were individually collected and lysed in 10 μl of RLT buffer DNA was purified using solid phase reversible immobilization (SPRI) beads and converted with bisulphite reagent using EZ DNA Methylation-Gold kit (ZYMO Research, D5005). DNA was eluted in EB buffer and individual blastocyst PBAT libraries were prepared as outlined below. First-strand synthesis was performed using Klenow exo-tagged (New England Biolabs) and a biotinylated oligo I (5-[Btn]CTACACGACGCTCTTCCGATCTNNNNNNNNN-3), followed by Exo-I treatment (New England Biolabs) and SPRI purification. Purified DNA was incubated with Dynabeads Strepatavidin M-280 beads (ThermoFisher Scientific) to capture biotinylated DNA and second strand synthesis was carried out using reverse oligo II (5'-TGCTGAACCGCTCTTCCGATCTNNNNNNNNN 3'). Libraries were amplified using indexed iPCRTag reverse primers (54) with KAPA HiFi (Kapa Biosystem) DNA polymerase for 15 PCR cycles and purified using SPRI beads (Agencourt Ampure XP bead). Libraries were quantitated and quality control was checked using Agilent High Sensitivity DNA kit and KAPA Illumina Library Quantifcation Kit for Illumina (KAPA Biosystems) Bisulfite-Seq (PBAT)