title: GSM5554049: SBR-84h_B; Mus musculus; RNA-Seq
library_construction_protocol: Gastruloids (24h: 240 gastruloids; 36h: 96 gastruloids; 48h: 48 gastruloids; 60h: 48 gastruloids; 72h: 24 gastruloids; 84h: 2x 24 gastruloids) were dissociated with Accutase and per sample 12,000 alive cells were sorted by FACS. Cellular supensions were loaded on a 10x Genomics Chromium Single Cell instrument. Single cell RNAseq libraries were prepared using the 10x Genomics Single Cell 3' Gel Bead and Library Kit according to CG000183 Single Cell 3' Reagent Kit v3 User Guide_RevA. GEMRT was performed in a Bio-Rad PTC-200 Thermal Cycler with 0.2ml PCR Tube Strips (Eppendorf P/N 0030 124.359): 53 °C for 45 minutes, 85 °C for 5 minutes; held at 4 °C. After RT, GEMs were broken and the single strand cDNA was cleaned up with DynaBeads® MyOneTM Silane Beads (Life Technologies P/N 37002D). cDNA was amplified using a Bio-Rad PTC-200 Thermal cycler with 0.2ml PCR Tube Strips (Eppendorf P/N 0030 124.359): 98 °C for 3 min; cycled 11x: 98 °C for 15 s, 63 °C for 20 s, and 72 °C for 1 min; 72 °C for 1 min; held at 4 °C. Amplified cDNA product was cleaned up with the SPRIselect Reagent Kit (0.6X SPRI). Indexed sequencing libraries were constructed using the reagents in the Chromium Single Cell 3' library kit V3 ( 10x Genomics P/N1000078), following these steps: 1) Fragmentation, End Repair and A-Tailing; 2) Post Fragmentation, End Repair & A-Tailing Double Sided Size Selection with SPRIselect Reagent Kit (0.6X SPRI and 0.8X SPRI); 3) adaptor ligation; 4) post-ligation cleanups with SPRIselect (0.8X SPRI); 5) sample index PCR using the Chromium Multiplex kit (10x Genomics P/N-120262); 6) Post Sample Index Double Sided Size Selection- with SPRIselect Reagent Kit (0.6X SPRI and 0.8X SPRI). The barcode sequencing libraries were quantified using a Qubit 2.0 with a Qubit TM dsDNA HS Assay Kit (Invitrogen P/N Q32854) and the quality of the libraries were performed on a 2100 Bioanalyzer from Agilent using an Agilent High Sensitivity DNA kit (Agilent P/N 5067-4626). Sequencing libraries were loaded at 1.6pM on an Illumina Nextseq500 with 75cycle kits using the following read length: 28 cycles Read1, 8 cycles i7 Index and 56 cycles Read2.