Tapestation high sensitivity d1000 kit

After 48 hours of in vitro culture, embryos were assessed morphologically. Analysis showed that 90.0% (36/40) of the embryos developed to the blastocyst stage and only 36.1% (13/36) of embryos reached the hatching stage. Representative images of the blastocysts in the hatching group and pre-hatching group are shown in Supplementary Fig 1 . Overall, 8 hatching blastocysts and 8 pre-hatching expanded blastocysts were randomly collected for subsequent RNA sequencing experiments. Whole single blastocysts were lysed in 2.3 µl of lysis buffer (0.8% (vol/vol), Triton X-100, and 2 U/μl of RNase inhibitor (both from Thermo Fisher, UK)) for cDNA synthesis using the SmartSeq2 method, as described previously (32) . Libraries were then prepared using using Nextera-XT (Illumine, US). ERCC RNA Spike-in mix (ThermoFisher, UK) was added at a 1/100,000 dilution. Amplified libraries were analysed for size distribution using the TapeStation High Sensitivity D1000 kit (Agilent, US). Libraries were then quantified using PicoGreen (Invitrogen, US) and relative volumes were pooled accordingly. Sequencing was then performed as 75bp paired-end reads on a HiSeq4000 system according to Illumina specifications.