GSM9450572: m18 Padi6 mutant blastocyst; Mus musculus; Bisulfite-Seq
DNA and RNA were separated using the G&T protocol [59]. Magnetic beads (MyOne C1, Life Technologies) were washed and annealed to oligo dTs and then used to capture polyadenylated mRNA from individual cell lysates. The supernatant containing the DNA was transferred to a new tube, and the beads washed three times in 1× FSS buffer (SuperScript II, Invitrogen), 10 mM DTT, 0.005% Tween-20 (Sigma), and 0.4 U/μl RNasin (Promega) to remove all DNA residues. Each washing solution was added to the DNA tube to maximize recovery. The single blastocyst BS-seq libraries were generated as described [60]. The DNA was purified from the lysate using a 0.9:1 ratio of Ampure XP beads (Beckman Coulter) and eluted into 10 μl of water. DNA purified from single blastocysts was treated with the EZ Methylation Direct kit (Zymo) for bisulfite conversion following the manufacturer's instructions. For the first-strand synthesis, the bisulfite-treated DNA was mixed with 40 μl of first-strand synthesis master mix (1× blue buffer [Enzymatics], 0.4 mM dNTP mix [Roche], 0.4 μM 6NF oligo), heated for 2 min to 65°C, and cooled on ice, followed by the addition of 50 U of exo− Klenow fragment DNA polymerase (Enzymatics) and incubation for 90 min at 37°C after slowly ramping up from 4°C. Exonuclease digestion was carried out by adding 20 U of exonuclease I (NEB) in a total volume of 100 μl for 1 h at 37°C. The resulting samples were purified using AMPure XP beads with a 0.8:1 ratio. The beads were mixed with 50 μl of second-strand master mix (1× blue buffer [Enzymatics], 0.4 mM dNTP mix [Roche], 0.4 μM 6NR oligo). The mixture was heated for 1 min to 98°C and then cooled on ice. Next, 50 U of exo− Klenow was added. The mixture was incubated on a thermocycler for 90 min at 37°C after slowly ramping up from 4°C. The resulting samples were purified using a 0.8:1 ratio of AMPure XP beads, and libraries were amplified using 50 μl of PCR master mix (1× KAPA HiFi Readymix, 0.2 μM PE1.0 primer, 0.2 μM iTAG index primer). The amplification process involved a 2-min step at 95°C, followed by 20 cycles of 80 sec at 94°C, 30 sec at 65°C, and 30 sec at 72°C, with a final extension of 3 min at 72°C. Finally, the libraries were subjected to purification using a 0.7:1 ratio of AMPure XP beads. After purification, the libraries were eluted in 15 µl of water, pooled together, and sequenced. The sequencing process involved the generation of pools of 20 libraries, which were sequenced on an Illumina NextSeq 500 platform. On average, the libraries were sequenced to generate 13.0 million paired-end reads with a read length of 75 bp.