title: GSM7159421: RNAseq_aCSF_Mg_R1; Mus musculus; RNA-Seq
library_construction_protocol: 24 h after injection, two mice were pooled per replicate and microglia were isolated from 1 mm tissue blocks spanning the injection sites. Tissues were incubated in lysis buffer (1 mg mL-1 collagenase D (Sigma-Aldrich), 0.05 mg mL-1 DNase1 (Sigma-Aldrich), 3 mM actinomycin D (ActD) diluted in Dulbecco's phosphate-buffered saline (DPBS) with Ca2+ Mg2+ (Thermo Fisher Scientific)) for 30 min at 37°C. Myelin was depleted using the debris removal kit (Miltenyi Biotec). Cells were treated with Fc-block in FACS buffer (DPBS supplemented with 0.2% BSA, Thermo Fisher Scientific) for 5 min at 4°C, then incubated with primary antibodies for 30 min at 4°C. The following primary antibodies were used at 1:200 dilution and purchased from Biolegend: CD11b (M1/70) and CD45 (30-F11). 15,000 to 20,000 CD45loCD11b+ microglia were FACS sorted into tubes containing RLT plus lysis buffer (Qiagen) supplemented with 1% 2-mercaptoethanol and 0.25% reagent DX (Qiagen) using FACSAria II with BD FACSDiva v8 software. Cells were first gated on SSC-A and FSC-A size and then doublet discrimination was performed with FSC-H and FSC-W parameters. Microglia lysates were frozen and stored at -80°C until processed for RNA-seq. Samples from plasma injection experiment were processed for RNA-seq using the Ovation RNA-seq System V2 low input kit (NuGEN). Libraries were equimolar pooled and sequenced on NovaSeq6000 S4 with 200 paired-end (Illumnia) to a depth of >50 million reads per library.