title: GSM6670812: RNAseq_ESC_Dux_KO_PlaB_rep2; Mus musculus; RNA-Seq
library_construction_protocol: For RNA-seq of embryos, 30 middle-2C embryos were collected 20 hours after microinjection in each independent biological replicates. Zona pellucida was removed by treating the embryos with Acidic Tyrode's solution. For RNA-seq of culture cells, total RNA was prepared using RNeasy Kit according to the manufacturer's instruction. The RNA solution was subjected to TURBO DNase treatment according to the manufacturer's instructions to remove genomic DNA carryover. For CUT&RUN, 1×105 culture cells freshly prepared by trypsin digestion were used to perform CUT&RUN assay with CUT&RUN Assay Kit (Cell Signaling Technology, 86652) according to the manufacturer's instructions. For RNA-seq of embryos, libraries were constructed with SMART-Seq Stranded Kit (TaKaRa, 634442) and indexed with SMARTer RNA Unique Dual Index Kit (TaKaRa, 634451) according to the manufacturer's instructions. For RNA-seq of culture cells, libraries were constructed using 1µg total RNA with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760L) and indexed with NEBNext Multiplex Oligos for Illumina (NEB, E6440S) according to the manufacturer's instructions. For CUT&RUN, enriched DNAs were subjected to library construction using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) with the adapter ligation step performed at 50°C instead of 65°C to prevent denature of small DNA inserts. The libraries were indexed with NEBNext Multiplex Oligos for Illumina according to the manufacturer's instructions.