Exonuclease 1 treatment

Total RNA was purified from sorted cell populations using the RNeasy Micro Kit (Qiagen), and concentration was determined using the Quant-IT RiboGreen RNA assay kit (Thermo Fisher Scientific). First-strand cDNA synthesis was performed with a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) followed by preamplification of genes of interest using the Fluidigm PreAmp Master Mix (Fluidigm Europe B.V.) with 25 ng of total RNA and in accordance with the manufacturer’s instructions. Exon-spanning primers to amplify genes of interest were designed using Primer-Blast (see Table 2 for details). To increase sensitivity, genes of interest were preamplified by 12 cycles of PCR using pooled assays followed by exonuclease I treatment (New England Biolabs) to remove unincorporated primers. Final preamplified cDNA was diluted 1:5 in Tris-EDTA buffer. Gene expression analysis was performed using the Biomark HD system from Fluidigm (Fluidigm Europe B.V.) in accordance with the manufacturer’s instructions and standard settings. Data were analyzed using the Real-Time PCR Analysis Software (Fluidigm Europe B.V.), and the resulting cycle threshold values were normalized to Ppia to obtain delta CT values.

Genome-wide methylation maps in KDM1A- and KDM1B-deficient MII oocytes and controls were generated using the PBAT method (Miura et al. 2012 (link)) as adapted in Peat et al. (2014) (link). For each genotype, triplicate PBAT libraries were generated, each from ∼100 MII oocytes. Each sample was spiked with ∼5 pg of λ DNA to assess conversion efficiency, which was 98.7%–99.2% for CpGs. Briefly, cells were lysed in EB buffer (Qiagen) with 0.5% SDS (Sigma, 161-0418) and bisulfite-treated using the one-step modification procedure in the Imprint DNA modification kit (Sigma, MOD50-1KT). The resulting DNA was purified using the EZ DNA methylation direct kit (Zymo, D5020). First strand synthesis was performed using Klenow Exo⁻ (New England Biolabs, M0212S) and a custom streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs (bp) of random sequence (9N). This was followed by exonuclease I treatment (New England Biolabs, M0293S), purification using SPRI beads (1.8× ratio; Fisher Scientific, 09-981-123), and binding to biotin beads (Life Technologies, 11205D). Samples were then subjected to second strand synthesis, again using custom primer, followed by 10 cycles of library amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads.