title: GSM6165992: Dnmt3l KO, E7.5, ExE, 1 (RNA-seq); Mus musculus; RNA-Seq
library_construction_protocol: Natural timed matings between heterozygous females and males were used for Dnmt3l, Dnmt3a, Dnmt3b and Dnmt3a/3b KO strains to generate KO and WT embryos. E7.5 epiblast, extra-embryonic ectoderm (ExE) and ectoplacental cone (EPC) for each embryo were manually separated. Single E7.5 epiblast and ExE samples were individually frozen in 10µL of buffer RLT Plus (Qiagen) for RNA-seq or PBAT; the corresponding EPC was used for genotyping.  Samples were homogenized in 400µL of TRIzol (Invitrogen) and phase separated with 80µL of chloroform:isoamyl alcohol (Sigma), centrifuging at 4°C for 15 minutes.  The aqueous phase was combined with 1µL GlycoBlue and 300µL of cold isopropanol. The RNA was pelleted by centrifugation for 10 minutes at 4°C, washed once with 75% ethanol, dried and then resuspended in 5µL of ultra-pure water.  To each RNA sample, 20µL of lysis/binding buffer was immediately added, proceeding directly to a single round of oligo(dT)25 capture of mRNA, using Dynabeads mRNA DIRECT kit (Life Technologies), using previously described adaptations for low starting material. Using the SMARTer Stranded RNA-seq kit (Clontech), mRNA libraries were prepared as per the manufacturers' instructions with 15 amplification cycles. Libraries were quantified using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification (KAPA). Libraries were multiplexed and sequenced using 50bp single-end on the Illumina HiSeq 2500 RapidRun. Using the SMARTer Stranded RNA-seq kit (Clontech), mRNA libraries were prepared as per the manufacturers' instructions with 15 amplification cycles. Libraries were quantified using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification (KAPA). Libraries were multiplexed and sequenced using 50bp single-end on the Illumina HiSeq 2500 RapidRun.