Illumina HiSeq 4000 paired end sequencing; Single cell RNA-seq of in vitro cultured human embryos
Single cell RNA-seq of in vitro cultured human embryos
Cryopreserved day 5 or day 6 human blastocysts were received at the University of Cambridge and thawed using Kitazato thaw kit (VT802-2, Hunter Scientific) following the manufacturer's instructions. Briefly, the day before thawing, the TS solution was placed at 37°C and Global Total human embryo culture medium (HGGT-030, LifeGlobal group) was incubated at 37°C and 5% CO2 . Upon thawing human embryos were placed in 1mL of the pre-warmed TS solution for 1 minute. They were subsequently transferred to DS solution for 3 minutes, WS1 solution for 5 minutes and WS2 solution for 1 minute. All these incubations were done using reproplates (REPROPLATE, Hunter Scientific). Thawed embryos were incubated in pre-equilibrated Global Total human embryo culture medium under mineral oil (9305, Irvine Scientific) for 4 hours to allow recovery. Human embryo culture was done at 37°C 21% O2 and 5% CO2. 4 hours post-thawing zona pellucida was removed by briefly treating the embryos with acidic tyrode's solution (T1788, Sigma). Embryos were subsequently cultured in pre-equilibrated In vitro culture 1 (IVC1) culture medium (M11-25, Cell Guidance Systems). We introduced a modification to our original IVC1 recipe (Shahbazi et al, 2016) consisting in the addition of 50 ng/mL of Insulin Growth Factor 1 (IGF-1) (78078, STEMCELL Technologies), as this improved the preservation of epiblast and hypoblast cells in culture (data not shown). Embryos were cultured in 8 well μ-slides tissue culture treated (80826, Ibidi). Half-media changes with IVC1 supplemented with IGF-1 were done every 24 hours. Where indicated 3 μM PD0325901 (Stem Cell Institute, Cambridge) were added to the culture medium. We dissociated a total of 6 human embryos (3 at day 9 and 3 at day 11 of development). Embryos were detached from the slides using a thin needle. They were subsequently placed in 500 μL of Trypsin EDTA (25300054, Life Technologies) and incubated at 37°C for 10 minutes. A single cell suspension was obtained by pipetting the embryos using 50 μm tips (MXL3-IND-50, Origio).  RNA was converted into cDNA and amplified as part of the 10x v2 chemistry. Libraries were constructed using the 10x v2 chemistry