GSM5597143: Dppa2-4_ctrl-x-WT_mating1_WT-b; Mus musculus; RNA-Seq
[Sample collection protocol]  Embryos from the Dppa2/4m- were generated from natural matings between males and females of relevant genotypes. The zona pellucida was removed using Tyrode's solution (Sigma, T1788) Individual embryos were placed in in 2.5µl methyltransferase reaction mixture, according to the published protocol (Clark et al., 2018). mRNA was captured using Smart-seq2 oligo-dT pre-annealed to magnetic beads (MyOne C1, Invitrogen). The lysate containing the gDNA was transferred to a separate PCR plate, and the beads were washed 3 times in 15 ml FSS buffer (Superscript II, Invitrogen), 10 mM DTT, 0.005% Tween-20 (Sigma) and 0.5 U/ml of RNAsin (Promega). The beads were then resuspended in 10 ml of reverse transcriptase mastermix (100 U SuperScript II (Invitrogen), 10 U RNAsin (Promega), 1 X Superscript II First-Strand Buffer, 2.5 mM DTT (Invitrogen), 1M Betaine (Sigma), 9mM MgCl2 (Invitrogen), 1 mM Template-Switching Oligo (Exiqon), 1 mM dNTP mix (Roche)) and incubated on a thermocycler for 60 min at 42oC, followed by 30 min at 50oC and 10 min at 60oC. PCR was then performed by adding 11 ml of 2 X KAPA HiFi HotStart ReadyMix and 1 ml of 2 mM ISPCR primer, and cycling as follows: 98oC for 3 min, 15 cycles of 98oC for 15 sec, 67oC for 20 sec, 72oC for 6 min, and finally 72oC for 5 min.  cDNA was purified using a 1:1 volumetric ratio of Ampure Beads (Beckman Coulter) and eluted in 20 ml of water. Libraries were prepared from 100 to 400 pg of cDNA using Nextera XT Kit (Illumina), per manufacturer's instructions but with one-fifth volumes for each sample.