title: GSM9495527: mESC, Spontaneous differentiation in EBs, D3; Mus musculus; RNA-Seq
library_construction_protocol: Single cell RNA sequencing was performed with the BD Rhapsody HT single cell analysis system (BD Biosciences, Heidelberg, Germany) using adapted protocols. Wildtype murine embryonic stem cells E14TG2α were harvested in four consecutive passages to form embryoid bodies for spontaneous differentiation in the absence of LIF for three, five, and seven days (D3, D5, D7) generating a total of n = 12 samples. EBs of each biological replicate were collected in separate reservoir tanks, transferred to 50 mL Falcon tubes, and sedimented via gravity before carefully removing supernatant. EBs were resuspended in 2 mL of TrypLE solution and incubated with constant mixing at 37°C for 1-2 minutes to generate single cell suspensions. Cells from each replicate and time point were separately centrifuged 5 min at 400 g, washed once with PBS with 1% FBS (FACS buffer). From here on, all steps were conducted on ice. Custom BDTM Single Cell Multiplexing Kit (#626545; anti-mouse MHC-calls I antibody, direct labelling) was used for sample tagging and pooled processing according to the manufacturer´s protocol. Labelled cells from four replicates (each 2,5 x 105 cells) were pooled for each individual time point to three samples (D3, D5, D7) and washed thrice in 2 mL of FACS buffer, centrifuged at 400 g for 5 min, and finally resuspended in 620 μL cold sample buffer. Staining cells with viability markers was achieved by adding 3.1 μL of 2 mM Calcein AM and 3.1 μL of 0.3 mM DRAQ7™ to the cell suspension in cold sample buffer (37°C in the dark, 5 min). Thereafter, 10 µL cells were counted using BD Rhapsody scanner 10 μL in an INCYTO™ disposable hemocytometer with the remaining cells kept on ice and protected from light. BD Rhapsody 8 lane cartridge were then primed, loaded with cells and, thereafter, with BD Rhapsody™ Enhanced Cell Capture Beads. After cell lysis and release of polyadenylated mRNAs, BD Rhapsody™ Enhanced Cell Capture Beads were retrieved, washed, and further processed for library preparation. Library preparation: • Reverse transcription (BD RhapsodyTM cDNA kit #633773) • Library preparation using BD RhapsodyTM Whole Transcriptome Analysis Amplification kit (#633801), generating Illumina-compatible libraries.  cDNA library preparation and sequencing were performed by the group of Dr. Inti Alberto De La Rosa Velásquez of the Core Facility Genomics (Helmholtz Center Munich, Germany) on an Illumina NovaSeq X Plus sequencer in a 10B200 flow as paired-end reads.