title: GSM1548411: Control Zygotes; Mus musculus; Bisulfite-Seq
library_construction_protocol: Frozen zygotes were lysed by incubation in a 1% SDS solution containing 0.2 µg/µL Proteinase K (Sigma) for one hour at 37ºC. Bisulfite conversion was performed directly on the lysate using the Imprint DNA Modification kit (Sigma) according to the manufacturer’s 2-step protocol, with the modification that samples were incubated at 65ºC for 90min, 95ºC for 3min then 65ºC for 20min. After addition of 1 µg yeast carrier RNA (Qiagen), treated DNA was purified with the PureLink PCR Micro Kit (Invitrogen) according to the manufacturer’s instructions. Before elution of DNA, on-column desulfonation was performed by incubation with BD buffer (Qiagen) for 8min. A complementary strand was synthesised by combining 1X NEB2 buffer (NEB), 0.25 mM dNTP mix (Roche) and 0.8 µM biotin labelled random tetramer primer (biotin-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNN) with purified DNA. This mix was incubated at 65ºC for 5min then 4ºC for 5min, followed by addition of 1 U/µL Klenow 3’-5’ exo- (NEB) and incubation at 4ºC for 15min. The temperature was then raised to 37ºC by 1ºC/min, and samples were incubated at 37ºC for 90min. DNA was purified using a 1:1 ratio of AMPure XP beads (Agencourt) according to the supplier’s protocol. The biotin-labelled strand was captured with Dynabeads M280 Streptavidin beads (Invitrogen) by incubation at RT for 20min, then separated from the original bisulfite treated strand by denaturation with freshly prepared 0.1N NaOH. While still on the beads, DNA complementary to the biotin-labelled strand was synthesised in the same manner as described above but with a non-labelled random tetramer primer (CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNN). The beads were resuspended in a mixture of 1X ThermoPol Reaction Buffer (NEB), 0.25 mM dNTP mix and 0.16 U/µL Bst DNA Polymerase Large Fragment then incubated at 65ºC for 30min. To elute from the beads, DNA was resuspended in a reaction mix composed of 1X Phusion HF Buffer (NEB), 0.25 mM dNTP mix, 0.4 µM PE1.0 oligo (Illumina) and 0.04 U/µL Phusion High-Fidelity DNA Polymerase (NEB) then incubated at 94ºC for 5min, 55ºC for 10min and 72ºC for 30min. DNA was purified using a 1:1 ratio of AMPure XP beads (Agencourt) according to the supplier’s protocol. PCR amplification was performed to generate a suitable library concentration for sequencing. The PCR reaction mix was composed of 1X Phusion HF Buffer (NEB), 0.1 mM dNTP mix, 0.4 µM each PE1.0 and PE2.0 oligos (Illumina) and 0.02 U/µL Phusion High-Fidelity DNA Polymerase (NEB). Cycling was performed by initial denaturation at 98ºC for 30s, followed by cycles of 98ºC for 20s, 65ºC for 20s and 72ºC for 20s, then a final extension at 72ºC for 5min.