GSM8844183: Cortex, Single cell, Injured, Ancrod - biol rep 2; Mus musculus; RNA-Seq
Cortical lesion microdissection Injury sites were isolated 6 days after injury. Mice were anesthetized with Avertin and  transcardially perfused with ice–cold, oxygenated artificial cerebrospinal fluid. The meninges  were removed, and the injury site and surrounding spared tissue were excised in an approximate  5 mm by 5 mm square, to a depth of up to but not including the corpus callosum. Tissue dissociation All steps were  performed with pre–chilled solutions on ice or at 4°C unless otherwise noted. The tissue was  roughly chopped with a razor blade, resuspended in Cutting Solution, then centrifuged at 300g  for 2 min at room temperature (RT). After aspirating the supernatant, the sample was processed  using the Miltenyi Neural Tissue Dissociation Kit–P (cat #130–092–628), according to  manufacturer's guidelines. During the second enzyme digestion step, cells were triturated 10  times each in 1,000, 750, and 500 uM fire–polished Pasteur pipettes. After the final  centrifugation, supernatant was aspirated, and cells were resuspended in 1 mL Red Blood Cell  Lysis buffer and incubated at RT for 1 min. This was stopped with 5 mL MACS Buffer and  centrifuged at 300g for 5 min. The resulting single–cell pellet was then incubated with Miltenyi  Myelin Removal Beads II, according to manufacturer's guidelines. Cell–bead suspensions were  passed through an equilibrated LS column that was then washed with 2 mL of MACS buffer.  After centrifugation at 300g for 5 min and supernatant aspiration, cells were resuspended in 50 uL of FACS buffer and kept on ice until sequencing. Single–cell suspensions were checked for viability, density, and purity using AOPI Stain  imaged on an Nexcelom K2 Cellometer. Samples were diluted to ~1,000 cells/uL and had a  minimum viability of 80%. ~6,000 cells per sample were sequenced with ~100,000 mean reads  and ~13,000 median UMI counts per cell. Sample libraries were prepared with the 10X  Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index) and multiplexed on the  sequencer with Dual Index Kit TT (Set A), according to manufacturer's protocols. These  libraries were then sequenced using paired–end sequencing on eight channels of an Illumina  Novaseq X 10–billion–read flow cell with a 300–cycle kit.