GSM7152484: embryo,replication1, scRNAseq; Mus musculus; RNA-Seq
Specific tissues of E8.5 embryos were pooled together in a low DNA bind Eppendorf tube and dissociated into single cell in 200μl TrypLE Express (Thermo Fisher Scientific, 12604013) for 15 mins at 37°C with agitation every 5 mins. 1ml 1xPBS + 0.4%BSA was added into the digestive buffer to quench the reaction. Then cells were centrifuged at 1200rpm for 5 mins and resuspended in 30μl PBS. Single-cell RNA-seq libraries were prepared using 10× Genomics Chromium Single Cell 5′ Reagent Kits according to the manufacturer's instructions. Brieﬂy, dissociated cells were resuspended to a ﬁnal cell concentration of ~1500 cells/μL with more than 85% viability as determined by Trypan Blue Solution (Thermo Fisher Scientific, 15250061). Cells were subjected to single-cell RNA sequencing aiming for a target cell recovery of up to 20,000 sequenced cells per sequencing library. After the reverse transcription step, emulsions were broken and barcoded-cDNA was puriﬁed with Dynabeads, followed by PCR ampliﬁcation. Ampliﬁed cDNA was then used for 5′ gene expression library construction.