Cell Dissociation of Isolated Gastrulating Murine Embryos for Single-Cell Sequencing

Proceed to perform the cell dissociation of isolated murine embryos once the genotypes have been confirmed. Using a P200 pipette, add 50 microliters of DMEM with 10%FBS to a new 1.5 milliliter tube. After pooling embryos with the same genotype into the new tube, place it on ice.

Allow the pooled embryos to settle to the bottom of the tube, then wash the embryos using 50 microliters of DPBS and wait for them to settle before removing as much DPBS as possible without removing the embryos. Next, add 100 microliters of trypsin to the pooled embryos and incubate at 37 degrees Celsius in a heat block for five minutes. Gently flick the 1.5 milliliter tube every 30 seconds to help the cells dissociate.

After five minutes, neutralize the trypsin with 300 microliters of DMEM containing 10%FBS. Centrifuge the embryos at 100 G for four minutes at room temperature. Resuspend the obtained small pellet in 40 microliters of DMEM with 10%FBS and place the tube on ice.

Mix five microliters of the cell suspension with five microliters of trypan blue in a new 1.5 milliliter tube, and pipette the mixture up and down thoroughly. Transfer the mixture onto a slide to determine the cell number and viability using an automated cell counter. Finally, proceed to single cell partitioning using a microfluidic chip, following the manufacturer's protocol.