For scMST, samples were collected on Whatman filter papers, fixed in 4% PFA overnight, and washed 3 times with PBS-0.2% triton, dehydrated in ethanol and stored in −80°C. The embryos we incubated through a sucrose gradient (5% 30 min, 15% 4h at 4°C and embedded into OCT as previously described 6 (link),7 (link) and 20 μm thick transverse cryosections were cut from the midbrain level onto silane coated coverslips.
For bulk RNAseq experiments, embryos were collected on Whatman filter papers in PBS and midbrain level neural plate border and specified neural crest cells, respectively, from left and right sides were manually microdissected using tungsten needles, immediately placed into RNA Lysis Buffer (RNAqueous™-4PCR Total RNA Isolation Kit, Thermofisher, Waltham, MA, USA) and stored at −80 °C. Replicates consisted of pooled tissue from at least five to seven embryos. Four replicates were collected per stage. For HH5 and HH6 replicates, rectangular patches lateral to the primitive streak were collected according to midbrain neural crest fate mapping as described 13 (link). For premigratory stages beyond HH6, neural plate borders or neural fold apices corresponding to the midbrain neural crest domain were excised. Beyond 7ss, dorsal neural tubes containing neural crest from the midline to migratory front were collected.
The scRNAseq samples were collected by dissecting midbrain slices (covering all germ layers) from the desired stage by using micro scissors, which were pooled from three to four embryos per sample and dissociated with a multi tissue dissociation kit (kit 3,130-110-204, Miltenyi Biotec,) at 37°C for 20 min with intermittent pipetting to achieve a single cell suspension.
For bulk RNAseq experiments, embryos were collected on Whatman filter papers in PBS and midbrain level neural plate border and specified neural crest cells, respectively, from left and right sides were manually microdissected using tungsten needles, immediately placed into RNA Lysis Buffer (RNAqueous™-4PCR Total RNA Isolation Kit, Thermofisher, Waltham, MA, USA) and stored at −80 °C. Replicates consisted of pooled tissue from at least five to seven embryos. Four replicates were collected per stage. For HH5 and HH6 replicates, rectangular patches lateral to the primitive streak were collected according to midbrain neural crest fate mapping as described 13 (link). For premigratory stages beyond HH6, neural plate borders or neural fold apices corresponding to the midbrain neural crest domain were excised. Beyond 7ss, dorsal neural tubes containing neural crest from the midline to migratory front were collected.
The scRNAseq samples were collected by dissecting midbrain slices (covering all germ layers) from the desired stage by using micro scissors, which were pooled from three to four embryos per sample and dissociated with a multi tissue dissociation kit (kit 3,130-110-204, Miltenyi Biotec,) at 37°C for 20 min with intermittent pipetting to achieve a single cell suspension.
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