GSM8636257: E22Q D14 1; Mus musculus; RNA-Seq
14 days after immunization, mice were euthanized via isoflurane overdose then perfused with chilled phosphate buffered saline. Lumbar spinal cords were manually dissected out and flash frozen immediately in liquid nitrogen then kept in liquid nitrogen. Lumbar tissue was later removed from liquid nitrogen and lysed in Nuclei Extraction Buffer (Miltenyi Biotec) with RNAse inhibitor (40U/mL RNasin Plus) in a gentleMACS tissue dissociator (Miltenyi) using octo coolers to keep nuclei chilled during digestion. Nuclei then were filtered over 100μM SmartStrainer into pre-chilled 5mL conicl tube. Nuclei were incubated with magnetic anti-nucleus microbeads (Miltenyi 130-132-997) for 15 minutes at 4c then were washed and passed onto LS magnetic column in cold room. Column was washed twice then nuclei were eluted and counted. RNase inhibitor was maintained in all buffers until nuclei were fixed. Nuclei were pelleted and resuspended in Fix/Perm Buffer (4% formaldehyde, 10X Genomics PN-2000517) and incubated overnight at 4c. The following morning fixation was quenched and then undewrent 10X Genomics Flex (Fixed RNA Profiling) protocol according to manufacturer recommendations including probe hybridization, washing (as individual samples), and then pooled and loaded onto a single lane of a Chip Q (10X Genomics PN-2000518) targeting 8000 nuclei per sample. Nuclei were partitioned into Gel Bead in Emulsion (GEMs) using a Chromium iX device (10X genomics). Libraries were prepared according to manufacturer's recommendation (10X Genomics "Chromium Fixed RNA Profiling Reagent Kits User Guide CG000527 Rev E") in which GEMs are incubated to allow for probe ligation and GEM barcoding, then libraries were amplified in bulk followed by size selection, sample index addition (using 9 amplification cycles), followed by a final size selection step. 1 NGS library was sequenced on a NovaSeq 6000 S4 flow cell in 1 lane (150bp paired end) with 10% PhiX spike-in at Novogene OTHER: single nucleus RNA templated ligation