RNA-seq of bulk trophectoderm microdissected from human blastocysts cultured to day 5, day 6 and day 7 post-fertilisation
RNA-seq of bulk trophectoderm microdissected from human blastocysts cultured to day 5, day 6 and day 7 post-fertilisation
Embryos were placed in drops of G-MOPS solution (Vitrolife; Cat. No. 10129) on a petri dish overlaid with mineral oil. The plate was placed on a microscope stage (Olympus IX70) and the embryos were held with an opposing holding pipette and blastomere biopsy pipette (Research Instruments) using micromanipulators (Narishige, Japan). The biopsy mode of a Saturn 5 laser (Research Instruments) was used to separate the mural TE from the ICM and polar TE. The separated mural TE was transferred to individual low bind RNAse-free tube containing 0.25 μl RNase inhibitor, 4.75 μl Dilution buffer (SMARTer Ultra Low Input RNA kit, Clontech; Cat. No. 634820) and 5 μl nuclease-free water on a pre-chilled CoolRack (Biocision, CA). Samples were stored at - 80°C until ready to be processed. cDNA was synthesized using SMARTer Ultra Low Input RNA for Illumina Sequencing-HV kit (Clontech Laboratories; Cat. No. 634820) according to the manufacturer's guidelines and as previously published (Blakeley et al., 2015b; Hyslop et al., 2016).  cDNA was sheared using Covaris S2 with the modified settings 10% duty, intensity 5, burst cycle 200 for 2 min. Libraries were prepared using Low Input Library Prep Kit (Clontech Laboratories; Cat. No. 634900) according to the manufacturer's instructions. Library quality was assessed with an Agilent 2100 BioAnalyser and concentration measured by QuBit broad range assay (Thermo Fisher; Cat. No. Q32850).