title: GSM5467830: run7_Gastruloid_F5_iX15; Mus musculus; RNA-Seq
library_construction_protocol: Cells were sorted into 384-well cell capture plates using a BD FACSAria III Cell Sorter (BD Biosciences) to collect live cells and sort only singlets. Plates were snap frozen on dry ice and stored at -80°C until further processing. All single cell libraries were prepared with the same conditions and reagents using the MARS-seq protocol as previously described (Jaitin et al, Science, 2014). All single cell libraries were prepared with the same conditions and reagents using the MARS-seq protocol as previously described (Jaitin et al, Science, 2014). Briefly, a Bravo Automated Liquid Handling Platform (Agilent) was used to reverse transcribe (Invitrogen #18080085) mRNA into cDNA with an oligonucleotide containing both the unique molecule identifiers (UMIs) and cell barcodes. Unused oligonucleotides were removed by Exonuclease I (New England Biolabs #M0293S) treatment. cDNAs were pooled (each pool containing half of a 384-well plate) for second strand synthesis (New England Biolabs #E6111S) and in vitro transcription amplification (New England Biolabs #E2040S). DNA template was removed (Invitrogen #AM2238) before fragmenting (Invitrogen #AM8740) and ligating (New England Biolabs #M0204S) resulting RNA to an oligo containing the pool barcode and Illumina sequences. Finally, RNA was reverse transcribed (Agilent Technologies #600107) and libraries were amplified (Roche #7958935001). Libraries were quantified with a Qubit 2.0 (Invitrogen) and their size distribution was determined by a 4200 TapeStation System (Agilent Technologies). Finally, libraries were pooled at equimolar concentration and sequenced on an Illumina NextSeq500, in 8 sequencing runs, using high-output 75 cycles v2.5 kits (Illumina #20024906). ScRNA-seq (MARS-seq)