title: GSM8296325: Olfactory bulb_EAE_replicate 3; Mus musculus; RNA-Seq
library_construction_protocol: Olfactory bulb tissue was harvested and snap-frozen in –80°C until further analysis. For nuclei isolation olfactory bulb tissues were homogenized in EZ lysis buffer (Sigma Aldrich, NUC101) with a glass douncer ((Sigma-Aldrich, D9063). After 3-min incubation on ice cell suspension was then centrifuged by 500 × g for 4 min at 4°C. Nuclei were washed once with the EZ lysis buffer and twice in the nucleus incubation buffer (MgCl2 2 mM, KCl 25 mM, Glycerophosphate 65 mM, Sucrose 340 mM, glycerol 5%, ethylenediaminetetraacetic acid 1 mM, bovine serum albumin (BSA) 1%) and filtered through a 30 µm pre-separation filter (Miltenyi Biotech, 130-041-407). The nuclei suspensions were stained in nucleus incubation buffer for ten minutes with anti-HuC/D labeled to Alexa647 (1:300, rabbit, Abcam; Cat# ab237235, RRID: AB_2864321) and propidium iodide (PI) (1:1000, Thermo Fisher Scientific, Cat# P1304MP). PI-positive HuC/D positive nuclei were sorted in PBS containing 0.4% BSA using a BD Aria III cell sorter (BD Biosciences). mRNA library preparation is performed according to the standard 10X Genomics protocol with the Chromium Next GEM Single Cell 3'GEM, Library & Gel Bead Kit v3.1 (Cat# 1000075, 10x Genomics). Briefly, gel bead emulsion (GEMs) was generated containing single nuclei and a master mix with reagents in the Chromium Controller followed by cell lysis and reverse transcription of RNA, cDNA amplification, enzymatic fragmentation, 5´adaptor attachment and sample indexing. On average around 25,000 nuclei were loaded on each channel of the Chromium instrument. To minimize batch effects, EAE and control samples were distributed evenly in the Chromium controller. Libraries were pooled and sequenced on an Illumina NovaSeq6000 platform (paired end sequencing, 2 x 100 bp read length, 500 million reads per sample).