title: GSM8830377: scRNA-seq (10X) of Day 4 e-stEM; Homo sapiens; RNA-Seq
library_construction_protocol: For scRNA-seq, 120h SACs were dissociated and sorted by flow cytometer for Smart-seq2 library construction. 60h SACs were collected after replating on gelatinized plate for 30min for feeder removal; day6 stEM and e-stEM were manually picked with a cut-tip, washed once with DPBS and then dissociated with TrypLE Express (Gibco) at 37 °C in 20%O2 and 5% CO2 incubator for 45-60 min. Library construction was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). For NovaSeq 6000 library construction, the process begins with RNA extraction and quality control, followed by mRNA enrichment using polyA selection or rRNA depletion. First and second strand cDNA synthesis is performed, then the cDNA is fragmented and end-repaired. Sequencing adapters are ligated to the cDNA fragments, which are then purified and size-selected. The library is amplified by PCR, assessed for quality, and loaded onto the NovaSeq 6000 for sequencing. For MGISEQ-2000, the protocol similarly starts with RNA extraction and quality control, mRNA enrichment or rRNA depletion, cDNA synthesis, fragmentation, and end-repair. Sequencing adapters are ligated, followed by purification, size selection, and PCR amplification. Unique to MGISEQ-2000 is a circularization step converting the linear library into a circular DNA molecule before loading and sequencing on the MGISEQ-2000 platform.