GSM3995485: Bin1-Homo-F-0333; Mus musculus; RNA-Seq
Microglia Isolation: Briefly, animals were anesthetized with Ketamine/Xylazine i.p. injection (1:1, concentration) and ice-cold PBS was used for perfusion and brains collection. After removing Cerebellum and Olfactory bulbs, remaining brain tissue was minced in Petri dish and transferred to 15ml tube with 3ml of Accutase (Cat: SCR005, Millipore); and incubated at 4°C for 30min in a rotary shaker. Then, large tissue chunks were allowed to settle at the bottom by gravity, and the cell suspension from the top of the tube were passed through pierce tissue strainer (Cat: 87791, Thermo Fisher) and collected in fresh tubes. Two ml of HBSS (Cat: 14185-052, Life Technologies)/HEPES (Cat: 15630-080, Life Technologies) buffer was added to the chunk tissues and triturated with 5ml pipette until almost homogenous. Following gravity separation, supernatants were passed through a pierce tissue strainer and pooled to the same tube containing cell suspension from previous step. Trituration was repeated with the remaining tissue chunks with a 1ml pipette until completely homogenous and passed through a pierce strainer and pooled to the rest of the cell suspension. Then, the strainer was rinsed with HBSS/HEPES buffer to bring the cell suspension to 15ml, prior to a spin at 600xg for 5min at 4°C (brake 5).  The supernatant was aspirated, and the pellet was resuspended with 1ml of 100% FBS. Nine milliliters of 33% Percoll (Cat: 17089102, GE Life Science) was added to the cell suspension and mixed well. One milliliter of 10%FBS was overlayed on the Percoll/cell suspension and spun at 800g at 4°C for 15min (break 1 or lowest possible). The supernatant was aspirated without disturbing the pellet, and the pellet was resuspended with 1ml of FACS buffer (HBSS, 1%BSA, 2mM EDTA, 25mM HEPES, 0.09% NaN3). Additional FACS buffer was added to bring the cell suspension to 15mL before spinning at 600g for 10min at 4°C. Following centrifugation, the pellet was resuspended with 300μl of FACS buffer and filtered through 30μm filter (Partec Celltrics, 04-004-2326) for further analysis. Microglia sorting: Filtered cells were pelleted down with gentle centrifugation and incubated with 50μl of Fc blocker anti-mouse CD16/32 (Cat: 101321, Biolegend) on ice for 10min. Then 50μl of CD11b-PE Rat anti-mouse antibody (Cat: 553311, BD Bioscience) and CD45 BV421 anti-mouse antibody (Cat: 563890, BD Bioscience) were mixed with the cells and incubated for 30min on ice. Following incubation, cells were washed and resuspended in 200μl of FACS buffer. Cells were filtered through a 30μm filter before FACS sorting (BD FACS Aria Fusion) and collecting approximately 20,000-100,000 CD11b+; CD45med cells in FACS buffer. Unstained and single-stained cells were used as controls for gating the cells in FACS. Sorted cells were centrifuged at 6800g for 15min and the cell pellets were snap frozen and stored at -80°C until RNA extraction. RNA extraction: Qiagen All Prep kit (Cat: 80224, Qiagen) was used for RNA extraction from previously frozen microglia cells. Briefly, cells were lysed with RLT Buffer (with β-mercaptoethanol added), and passed through QIA shredder (Cat: 79654, Qiagen). Flow-through from the QIA shredder was then passed through a DNA Mini column to remove the DNA from the lysate. DNA mini columns were stored at 4°C for DNA purification later; and the passed-through lysates were treated with proteinase K. Following treatment, 100% ethanol was added to the lysates and passed through an RNeasy Mini spin column. In-column DNase treatment was also performed. Subsequently, RNA was eluted with RNase-free water, quantified in Nano-drop and stored at -70°C until samples were submitted for sequencing. mRNA profile of  microglia cells FACS-sorted from brains of mice homozygous and heterozygous conditional knock-out for Bin1 in excitatory neurons  (B6.129S6-Bin1tm2Gcp/J (Bin1flox/flox), <FVB/N-Tg(Thy1-cre)1Vln/J> (Thy1-Cre) ) and Bin1-expressing littermates at 3 month of age had been generated from 10ng of total RNA using SMARTer Ultra Low Input RNA kit v4 (Clontech).  The cDNA was amplified by 8 PCR cycles, followed by QC analysis on BioAnalyzer 2100 (Agilent).  Sequence libraries were produced from 150pg of cDNA using Nextera XT DNA library kit (Illumina), cleaned up with AMPure XP beads, and QC checked with Caliper LabChip GX.