GSM7961715
Supernumerary human embryos donated to research by patients attending CReATe Fertility Centre, with study approval by Veritas IRB (protocol number 16580) and regional ethics board (2018/691-31 and 20.126) were utilised. Embryos were thawed using the Vit Kit-Thaw (FujiFilm). A total of 87 embryos were cultured from embryonic days 3-7 in a SAGE 1-step medium. Embryos were then dissociated into single cells as previously described using TrypLE enzyme solution and micromanipulation with glass capillaries. Cells were manually collected into individual 8-strip PCR tubes containing 2 uL of lysis buffer (0.13% Triton X-100, 1.33 U/ul Recombinant RNase inhibitor, 2.0 uM 5.8S rRNA-masking oligonucleotide) and stored at -80°C until further processing. To identify embryonic lineages based on transcriptomic signatures, single-cell mRNA-sncRNA Co-seq was performed as previously described and validated on mouse embryos. Briefly, individual cells were collected into 4uL of lysis buffer. After cell lysis at 72°C for 20 minutes, lysates were split into two equal 2 µL halves, with one half allocated to sncRNA sequencing and the other to SmartSeq. Each half was prepared following the original Small-seq and SmartSeq2 protocols, using 22 amplification cycles during the first PCR of SmartSeq2. This approach enabled the association of mRNA markers of cell lineage with corresponding sncRNA profiles. Small-seq Library Preparation and Sequencing:The remaining, non-Coseq sncRNA libraries were generated using the Small-seq protocol as previously described. Briefly, cells were thawed and incubated at 72°C for 20 minutes followed by 3' adapter ligation at 30°C for 6h followed by 4°C for 10h. Free adapters were then removed by 5' deadenylation followed by digestion of RT primer-3' adapter duplexes by lambda exonuclease. The 5' adapter was ligated at 37°C for 1h, followed by reverse transcription with Superscript II (ThermoFisher Scientific) at 42°C for 1h. Two PCRs were performed, first to amplify the cDNA products (12 cycles) and second to barcode the libraries for multiplexing (12 cycles). The resulting pooled libraries (~150 nt) were processed with the DNA Clean & Concentrator (Zymo) at a 7x buffer:DNA ratio, eluted in 50 uL, analysed by 2100 Bioanalyzer (Agilent) and quantified by Qubit DNA High Sensitivity (ThermoFisher Scientific). Libraries were sequenced on either NovaSeq or NextSeq 550 100 bp SE or PE for a sequencing depth of 2 million reads per cell. During the following analysis, only the forward read was used from PE data. Co-seq to capture sncRNA and mRNA from a single-cell: Briefly, individual cells were collected into 4uL of lysis buffer. After cell lysis at 72°C for 20 minutes, lysates were split into two equal 2 µL halves, with one half allocated to sncRNA sequencing and the other to SmartSeq. Each half was prepared following the original Small-seq and SmartSeq2 protocols, using 22 amplification cycles during the first PCR of SmartSeq2. This approach enabled the association of mRNA markers of cell lineage with corresponding sncRNA profiles.