GSM4974512: WT Middle stage antheridia rep2 (Smallwood BS seq); Marchantia polymorpha subsp. ruderalis; Bisulfite-Seq
Tak-1 thallus tissue was collected from 2-week old plants.Sperm were isolated and developing antheridia were manually dissected as described previously (Schmid et al., 2018; Higo et al., 2016). Embryos were dissected from fertilised gametophores 7 days following sperm application and the surrounding pseudoperianths were removed. DNA and RNA were extracted simultaneously from the frozen antheridium halves and sperm using the DynabeadsTM mRNA DIRECTTM Micro Kit (Invitrogen).  Thallus, sperm, and embryo DNA was extracted as described (Healey et al., 2014). For the thallus, embryo and sperm, single-end bisulphite-sequencing libraries for Illumina sequencing were constructed with Ovation Ultralow Methyl-Seq Library Systems (Nugen, 0336) and EpiTect Fast Bisulphite Conversion (Qiagen, 59802) kits according to manufacturer's instructions, with the incorporation of either one (WT and dn4mt mutant sperm) or two rounds (WT sperm, thallus, and embryo) of bisulphite conversion. For antheridia, WT sperm, and CMTa knockdown sperm, bisulphite sequencing library preparation was carried out as previously described (Picelli et al., 2014; Smallwood et al., 2014).