title: GSM8822417: uBT_SC_MG_4m_KO_3_LPS; Mus musculus; RNA-Seq
library_construction_protocol: Microglial isolation was performed as previously described (Hammond et al.). Mice were all females and were deeply anesthetized using xylazine (Rompun 0.2%) and ketamine (Imalgene 500) and transcardially perfused with ice-cold 0.1 M PBS and whole brains were collected. Brains were Dounce homogenized in ice cold Hank's balanced salt solution (HBSS) 10 times with the loose and 5 times with tight pestles. Tissues were passed through a 70-μm cell strainer (Corning). Cells were pelleted by centrifugation (300g, 5 minutes), resuspended in 40% Percoll solution (GE Healthcare), HBSS and centrifugated (30 minutes, 500g, 4°C). Myelin was then removed with the Percoll. Cell pellet was washed in HBSS and resuspended in PBS/2mM EDTA (Life Technologies).  Cells were stained with Zombie Green (1/1000, Biolegend) for 15 minutes at 4°C. All samples were then washed and resuspended in ice cold FACS buffer (5% FCS, 2mM EDTA, in 1x PBS) with TruStain FcX (Biolegend) for 10 minutes. Antibodies were then added Cd11b (PE, 1/100, Biolegend 101207), CD45 (SBB700, 1/20, Biorad, MCA1031) and incubated for 20 minutes at 4°C. Finally, cells were sorted using Biorad S3 cell sorter. Approximately 10000 to 30000 cells were isolated per sample and directly lysed with RNA lysis buffer (RNeasy micro kit, Qiagen). Lysates were stored at -80°C before RNA extraction according to manufacturer's protocol (RNeasy micro kit, Qiagen). Quantity of RNA used for sequencing was adjusted to the number of cells sorted, and 2500 to 5000 cells were sequenced. Since the RNAconcentration was too low to be detected using a Nanodrop, test samples were used to assess  RIN quality. RNA quantity sequenced was adjusted to the number of FAC-sorted cells, to sequence between 2500 and 5000 cells per sample.cDNA synthesis was performed using the Smartseq V4 TAKARA kit with linear pre-amplification. Library preparation was made with amplified cDNA for every sample using the Nextera XT DNA Library Preparation Kit (Illumina). 35 samples were run with a 1200 million cartridge to reach an average coverage of 34 million reads per sample (100bp, paired-ends). Sequencing was performed using the Novaseq 6000 sequencer (Illumina). Library preparations and sequencing were performed by the ICM iGenSeq core facility.