GSM7063540: RNA-seq_Microglia, SMAD4-conditional KO, Rep1; Mus musculus; RNA-Seq
Mouse brains were homogenized as previously described by gentle mechanical dissociation (Gosselin et al, 2014) and stained with anti-mouse anti-CD11b-APC (BioLegend 101212), anti-CD45-Alexa488 (BioLegend 103122), and anti-CX3CR1-PE (BioLegend 149006). Microglia were sorted as DAPI negative, singlets, and CD11b+CD45lowCX3CR1+. Total RNA was extracted from sorted cells using the Direct-zol RNA MicroPrep Kit (Zymo Research R2052) and stored at -80°C until library preparation. RNA-seq libraries were prepared as previously described (Sakai et al., 2019, Immunity)  with approximately 100,000 sorted live microglia. mRNAs were enriched from total RNA by incubation with Oligo d(T) Magnetic beads (NEB, S1419S) in 2X DTBB buffer (20 mM Tris-HCl pH 7.5, 1 M LiCl, 2mM EDTA, 1% lithium dodecyl sulfate, 0.1% Triton X-100) at 65°C for 2 minutes and were incubated at room temperature while rotating for 15 minutes. The beads were then washed 1x with RNA Wash Buffer 1 (10mM TrisHCl pH 7.5, 0.15M LiCl, 1mM EDTA, 0,1% lithium dodecyl sulfate 0.1% Triton X-100) and 1x with RNA Wash Buffer 3 (10mM Tris-HCl pH 7.5, 0.15 M NaCl, 1mM EDTA) before elution in RNA Elution Buffer (10mM Tris-HCl pH 7.5, 1mM EDTA) at 80°C for 2 minutes. PolyA selection was performed a second time, and samples were washed 1x with Wash Buffer 1, 1x with Wash Buffer 3, and 1x with 1x SuperScript III first-strand buffer. Beads were then resuspended in 10 µL 2x SuperScript III buffer plus 10 mM DTT, and RNA was fragmented at 94°C for 9 minutes and immediately chilled on ice before the next step. For first-strand synthesis, 10 µL of fragmented mRNA, 0.5 µL Random primers (50 µM)(Thermo Fisher), 0.5 µL SUPERase-In (Ambion), 1µL dNTPs (10mM), and 1µL of DTT (10mM) were heated for 50°C for one minute. At the end of incubation, 5.8 µL of water, 1 µL of DTT (100mM), 0.1 µL Actinomycin D (2µg/uL), 0.2uL of 1% Tween-20 (Sigma) and 0.5µL of SuperScript III (Thermo Fisher Scientific) were added and incubated in a PCR machine using the following conditions: 25°C for 10 minutes, 50°C for 50 minutes, and a 4°C hold. The product was then purified with RNAClean XP beads (Beckman Coulter) according to manufacturer's instruction and eluted with 10 µL nuclease-free wate. The RNA/cDNA double-stranded hybrid was then added to 1.5 µL Blue Buffer (Enzymatics), 1.1 µL of dUTP mix (10mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 µL RNase H (5U/uL), 1.05 µL of water, 1µL of DNA Polymerase I (Enzymatics) and 0.15 µL of 1% Tween-20. The mixture was incubated at 16°C overnight. The following day, the dUTP marked dsDNA was purified using 28 µL of SpeedBeads (GE Healthcare), diluted with 20% PEG8000, 2.5M NaCl to a final concentration of 13% PEG, eluted with 40 µL elution buffer (DNA elution buffer from Zymo ChIP Clean and Concentrator Kit). The purified dsDNA underwent end repair by blunting, A-tailing, and adaptor ligation as previously described 14 using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR amplified for 16 cycles, size for 200-500bp size range, quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and sequenced on a HiSeq 4000 for 51 cycles according to the manufacturer's instructions.