title: GSM2934655: 1102S7: EarlyDisease5; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized with ketamine/xylazine cocktail and perfused with ice-cold PBS. Spinal cords were flushed from the spinal column using PBS and placed in ice-cold HBSS with HEPES. Tissue was homogenized using Wheaton tissue grinders to achieve a single cell suspension. The homogenate was centrifuged through a 30% Percoll gradient to remove myelin and enrich for microglia. After washing with FACS buffer (HBSS, HEPES, BSA, EDTA) cells were Fc-blocked (CD16/32, Cat. #101321, Biolegend, San Diego, CA, USA) and stained with CD11b-PE (Cat. #553311, BD Biosciences, MA, USA) and CD45-BV421 (Cat. #103134, Biolegend). DRAQ7 was used for dead cell exclusion. Cells were sorted on an ARIA Fusion Flow Cytometer and collected directly into RNA lysis buffer. RNA was isolated with the RNeasy Micro Kit Plus from Qiagen. Libraries were constructed using the SMART-Seq Ultra Low-input (Clontech, Mountain View, CA, USA) and NexteraXT kits (Illumina, San Diego, CA, USA). Libraries were sequenced at an average depth of 20 million paired end reads.