title: GSM5530521: Irf8KO_withoutLaser_S3; Mus musculus; RNA-Seq
library_construction_protocol: Fluorescence-activated cell sorting of retinal microglia: Following intracardic perfusion with 1× PBS and enucleation, eyes were dissected in ice-cold 1× PBS to isolate the retinae. For the lasered mice, the central parts (70%) of the retinae were used for FACS while the peripheral parts were omitted. After tissue homogenization and filtration through a 50 µm cell strainer (Sysmex, Goerlitz, Germany), dead cell exclusion was performed by incubation with fixable viability dye 780 (1:1000, 65-0865-14, eBioscience, Waltham, MA, US). Anti-CD16/CD32 Fc block (1:200, 553142, BD Biosciences, Heidelberg, Germany) was used to avoid unspecific binding. Following staining against CD45 (1:200, 103133, BioLegend, San Diego, CA, US), CD11b (1:200, 17-0112-83, eBioscience, Waltham, MA, US), Ly6C (1:200, 560593, BD Bioscience, Heidelberg, Germany) and Ly6G (1:200, 560601, BD Biosciences, Heidelberg, Germany) for 20 minutes at 4 °C, retinal microglia characterized as CD45lowCD11b+Cx3cr1GFP/+Ly6C-Ly6G- were analyzed and sorted into RNA stabilization reagent (QIAGEN, Hilden, Germany) using a MoFlo Astrios EQ High Speed Cell Sorter (Beckman Coulter, Munich, Germany). For RNA Sequencing, an average of 10,000 retinal microglia per sample were obtained from pooling 3 to 4 mice. RNA extraction: RNA extraction, RNA library preparation and RNA sequencing were performed in collaboration with the Genomics Core Facility "KFB-Center of Excellence for Fluorescent Bioanalytics" (University of Regensburg, Germany). RNA extraction was performed according to manufacturer's instructions using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). After pelleting the sample by centrifugation, the RNA stabilization reagent was removed and replaced by RLT Plus buffer for lysing of retinal microglia. Genomic DNA was removed selectively and efficiently by using gDNA Eliminator spin columns for RNA purification. After adding Ethanol to the flow-through, the sample was applied to an RNeasy MinElute spin column to collect RNA. Finally, after washing of the column, all purified RNA was eluted in RNase-free water. The quality and integrity of total RNA was assessed with a Agilent 2100 Bioanalyzer in combination with the RNA 6000 Pico LabChip Kit (Agilent, Palo Alto, CA, USA). RNA sequencing: First-strand cDNA was generated using SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, inc., Mountain View, CA, USA). Double-stand cDNA was amplified with LD PCR and purified with AMPure XP beads. Library preparation was constructed conforming to the Illumina Nextera XT Sample Preparation Guide (Illumina, San Diego, CA, USA). In brief, 150 pg of input cDNA was tagmented via Nextera XT transposome. The products were purified and amplified with a limited-cycle PCR program ro construct sequencing libraries. The libraries were quantified with the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Wobum, MA, USA). Equimolar amounts of each library were pooled for cluster generation on the cBot using the Illumina TruSe SR Cluster Kit v3. The sequencing run was performed on a HiSeq1000 instrument with TruSeq SBS Kit v3 according to the Illumina HiSeq 1000 System User Guide. Illumina image analysis and base calling were recorded in library base call format (.bcl) and further converted to Fastq files via the CASAVA1.8.2 software.