GSM7864592: Grhl3-null replicate 2; Mus musculus; RNA-Seq
To generate animals for scRNA-seq, Grhl3+/- mice were intercrossed through timed mating. E8.5 embryos were harvested at 3 p.m. on the eighth day post-fertilisation. Embryos were dissected in DEPC-PBS and the yolk sac samples were utilised for genotyping as described above. The caudal half of five wild-type and three Grhl3-null embryos was separated using tungsten needles in 0.04% BSA (Sigma-Aldrich) in DEPC-PBS and then pelleted through centrifugation at 300 rcf for 3 mins at 4℃. The supernatant was removed, and the caudal half of the embryo was then incubated with 50 μL 0.25% (v/v) trypsin (Gibco) in DEPC-PBS for 5 mins at 37℃ and triturated with a 200 μL pipette tip to dissociate tissues. The digestion reaction was then inactivated by adding 100 μL 10% (v/v) FBS (Bovogen Biologicals) in DMEM (Gibco). Cells from five wild-type embryos and from three Grhl3-null embryos were pooled and filtered through Flowmi Cell Strainers (40 μm). Cells were then pelleted through centrifugation at 300 rcf for 3 mins at 4℃ and resuspended in 15 μL 10% (v/v) FBS (Bovogen Biologicals) in DMEM (Gibco) per embryo. Cell number and viability were assessed using a hemocytometer by mixing 8 μl cell suspension with 8 μl 0.4% (v/v) Trypan Blue Solution (Sigma-Aldrich) and a ~95% cell viability was observed in both wild-type and Grhl3-null samples. Cells were then diluted in 10% (v/v) FBS (Bovogen Biologicals) in DMEM (Gibco) to a final concentration of 1,000 cells/μL for single cell droplet generation and library preparation. A Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 and a Chromium Controller from 10X Genomics were used to generate single cell droplets as per manufacturer's instructions. Two wild-type and two Grhl3-null reactions were performed. Briefly, cell suspension with a master mix, gel beads and partitioning oil were loaded onto a Chromium Chip B to generate Gel Beads-in-emulsion. After this, samples were transferred into PCR tube strips and proceeded for reverse transcription and template switching using a T100 Thermal Cycler (Bio-Rad Laboratories). Synthesised full-length cDNA was then purified and amplified, followed by a clean-up reaction. The quality and quantity of amplified cDNA were assessed using an Agilent 4200 TapeStation with a High Sensitivity D5000 ScreenTape. Two wild-type and two Grhl3-null 3' gene expression libraries were then constructed for sequencing as outlined in the manufacturer's user guide. In short, cDNA amplicon from the previous reaction was fragmented and ligated with adaptors, and then subjected for a sample index PCR and a size selection with SPRIselect reagent. The quality and quantity of the libraries were again assessed again using an Agilent 4200 TapeStation with a High Sensitivity D5000 ScreenTape. Finally, the libraries were sequenced on an Illumina NextSeq 500 System with the NextSeq 500/550 High Output Kit v2.5 at a targeted sequencing depth of 400 million reads per library.