GSM8835987: E16.5 cervical spinal cord, replicate 4, chromium reaction 2 of 2; Mus musculus; RNA-Seq
Female wild-type mice with a pregnancy dated to embryonic day E14.5 or E16.5 were used. Each experimental replicate consisted of a pooled litter of 3-10 spinal cords taken from embryos of one single pregnant female. The expediency of the experiment did not allow sex-typing of embryos prior to pooling and, as such, all biological replicates are a mix of sexes. Embryos were rapidly dissected and immersed in cold 1X PBS. Spinal cords were sequentially dissected as described above and placed in cold dissection medium (50 ml HBSS-calcium/magnesium-free with phenol red (Gibco) + 750 µl HEPES (1M, Gibco)). When possible, extra care was taken to remove meninges and dorsal root ganglia from the isolated cord. From here, appropriate spinal cord segments were dissected from the cord, and then cut into fine pieces with dissecting spring scissors. Either cervical, thoracic or lumbosacral cords were isolated based on the experimental group. In either case, care was taken not to include transitional zones of the cord (e.g. where cervical tapers into thoracic or where thoracic begins to enlarge to lumbar); due to this, these regions are likely underrepresented in our data. Pooled spinal cord tissues were then added to a 15 ml conical tube containing dissociation medium (100 units Papain (Worthington), 4.75 ml HBSS+CM (calcium and magnesium; Gibco), and 250 µl of DNAse (Worthington, 1000 units resuspended in 500 µl of HBSS+CM)). The tube was gently inverted several times every 5 minutes while leaving the tube to incubate at 37 °C between inversions. After 20 minutes, the suspension was triturated gently with a series of three fire-polished glass pipettes (Fisher Scientific) with progressively finer bores. Suspensions are then centrifuged for 8 minutes at 850 rpm; after this, the supernatant was removed and the cells were gently resuspended with a solution of 4.5 ml HBSS+CM, 600 µl ovomucoid protease inhibitor solution (Worthington, resuspended in 30 ml HBSS+CM), and 250 µl DNAse (as above). Debris is allowed to settle for 10 minutes, at which point the supernatant with suspended cells was added to a second 15 ml conical tube containing neutralizing solution (10 ml HBSS+CM + 100 mg Trypsin inhibitor (Sigma) + 100 mg bovine serum albumin (Sigma)). This suspension was centrifuged once more for 8 minutes at 850 rpm, and the supernatant was removed. The remaining cells were resuspended in approximately 1 ml of 1X PBS containing 5 µl of molecular-grade BSA (New England Biolabs) and 2 ul of RNAse-inhibitor (Biosearch Technologies). Cell concentration in this suspension was assessed using a standard hemacytometer (a 50:50 sample of cell suspension + Trypan blue). The cell suspension was diluted with the same resuspension medium until the final concentration was appropriate for loading into the 10X Genomics Chromium chip. Produced based on manufacturer's instructions (10X Chromium 3' gene expression user guide; CG000204 Rev D)