GSM8463162: B6_Nx_BMDM_rep2; Mus musculus; RNA-Seq
Mice were euthanized with CO2. After respiratory movements have ceased, mice were transferred to a dissection area, the abdomen and chest were opened through a midline incision. The aorta was cut and the lungs were perfused with ice cold PBS through the right ventricle until the lungs were cleared of blood. Lungs were gently removed from the thorax, the lobes were dissected from the trachea, placed in sterile tubes and snap frozen in liquid nitrogen. Tissue was stored at -80°C until used for downstream RNA-seq, ChIP-seq analysis and protein measurements. Total RNA was isolated from lung tissue or culture cells and purified using a Direct-zol RNA MicroPrep Kit (Zymo Research) as described by the manufacture.  mRNAs were enriched by incubation with Oligo d(T) Magnetic Beads (NEB).  For ChIP-seq, frozen mouse lung tissues were pulverized on dry ice and the tissue powder was cross-linked with 2 mM disuccinimidyl glutarate (DSG) (ProteoChem) in PBS for 30 min at room temperature, and then directly a second crosslinking was performed by the addition of 1% (vol/vol) formaldehyde in PBS for 10 min. The crosslinking reaction was quenched by 0.125 M glycine (Sigma-Aldrich). The tissue powder was washed once with ice-cold PBS and pelleted at 1000 g for 5 min at 4°C. Crosslinked cells were resuspended in 100 l of PIPA-NR buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.4% sodium deoxycholate, 0.1% SDS, 1% NP-40, 0.5 mM DTT, 1 mM PMSF, 0.5 mM sodium butyrate, 1X protease inhibitor cocktail). Chromatin DNA was sonicated in a 96-well plate (Costar) using PIXUL™ Multi-Sample Sonicator (Active Motif) with the following setting: Process Time, 90 min; Pulse, 50 N; PRF, 1.00 kHz; Burst Rate, 20 Hz. Samples were centrifuged at 15000 rpm for 10 min at 4C, and the supernatant was used for immunoprecipitation. For RNA-seq, to fragment Poly A-enriched mRNA, mRNAs were incubated in 2X Superscript III first-strand buffer (Invitrogen) with 10 mM DTT at 94°C for 9 minutes.  The 10 µ l of fragmented mRNAs were incubated with 0.5 µl of Random primers (3 µg/µl) (Invitrogen), 0.5 µl of Oligo dT primer (50 µM) (Invitrogen), 0.5 µl of SUPERase-In (Ambion), and 1 µl of dNTPs (Invitrogen) at 50°C for 1 min.  After the incubation, 5.8 µl of water, 1 µl of DTT (100 mM), 0.1 µl of Actinomycin D (2 µg/µL), 0.2 µl of 1% Tween-20 and 0.5 µl of Superscript III (Invitrogen) were added and incubated on the following conditions: 25°C for 10 min, 50°C for 50 min, and 4°C hold.  The mixture was purified with RNAClean XP beads (Beckman Coulter) as described by the manufacture and eluted with 10 µl of water.  For second-strand synthesis with dUTP, the RNA/cDNA double-stranded hybrid was then added to 1.5 µl of Blue Buffer (Enzymatics), 1.1 µl of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 µl of RNAse H (5 U/µl) (Enzymatics), 1.05 µl of water, 1 µl of DNA polymerase I (Enzymatics), and 0.15 µl of 1% Tween-20.  The mixture was incubated at 16°C for 2.5 hr.  The resulting dUTP-marked dsDNA was purified with 2 µl of SpeedBeads (GE Healthcare) in 20% PEG 8000/2.5M NaCl (final 13% PEG 8000), and eluted with 40 µl of EB (Zymo Research).  The eluted dsDNA was carried out end repair by blunting, A-tailing and adapter ligation as previously described (Heinz et al., 2010) using barcoded adapters (Bioo Scientific).  The end repaired dsDNA was amplified for 16 cycles using 0.05 U/µl KAPA High Fidelity HotStart (Kapa Biosystems) and 1 µM each of primers Solexa 1GA and Solexa 1GB.  The amplified libraries were selected using PAGE/TBE gel (Invitrogen) for 225-325 bp fragments by gel extraction. Chromatin immunoprecipitation (ChIP) was performed as previously described (Heinz et al., 2018).