title: Illumina HiSeq 2500 paired end sequencing; A spatially resolved single cell atlas of human gastrulation
design: A spatially resolved single cell atlas of human gastrulation
library_construction_protocol: The sample was collected in L15 media before briefly being transferred to M2 media (Sigma-Aldrich, Cat No. M7167) for dissection and then dissociated into single cells using 200μl Accutase (ThermoFisher, Cat No. A1110501) for 12 minutes at 37°C, being agitated every 2 minutes, before adding 200μl heat-inactivated FBS (ThermoFisher, Cat No. 10500) to quench the reaction. Cells were then centrifuged at 1000rpm for 3 minutes at 4°C before being suspended in 100μl HBSS (ThermoFisher, Cat No. 14025) + 1% FBS, and stored on ice. Single cells were collected using a Sony SH800 FACS machine with a stringent single-cell collection protocol and sorted into 384 well plates containing SMART-seq2 lysis buffer plus ERCC spike-ins. To ensure good quality cells were collected, a live/dead dye (Abcam, Cat No. ab115347) was used; 100μl was added to the cell suspension at a 2x concentration in HBSS 10 minutes before collection, and live cells were collected based on their FITC intensity. Once cells were collected, plates were sealed, spun down, and frozen using dry ice before being stored at -80°C. This complete process, from dissection to single-cell collection, took approximately 2-3 hours. mRNA from the single embryo was extracted and amplified using the SMARTSeq2 protocol, with the additional inclusion of ERCC spike-in control.  Multiplexed sequencing libraries were generated from cDNA using the Illumina Nextera XT protocol.