GSM7063507: Microglia, SALL1 ChIPseq Input, SALL1 Enhancer WT, Rep2; Mus musculus; ChIP-Seq
Mouse brains were homogenized as previously described (Gosselin et al. 2014)  by gentle mechanical dissociation. Cells were then incubated in staining buffer on ice with anti-CD16/32 blocking antibody (BioLegend 101319) for 15 minutes, and then with anti-mouse anti-CD11b-APC (BioLegend 101212), anti-CD45-Alexa488 (BioLegend 103122), and anti-CX3CR1-PE (BioLegend 149006) for 25 minutes. Cell preparations for H3K27ac ChIP-seq were fixed with 1% formaldehyde for 10 minutes and quenched with 0.125M glycine for 5 minutes after staining, and subsequently washed three times. Cells were washed once and filtered through a 40 M cell strainer. Sorting was performed on a Sony MA900 or MoFlo Astrios EQ cell sorter. Microglia were defined as events that were DAPI negative, singlets, and CD11b+CD45lowCX3CR1+. Sorted microglia were pelleted at 800xg for 5 minutes, pellets were then snap frozen and stored at -80°C prior to library preparation. Brain nuclei were isolated as previously described (Nott et al., 2019), with initial homogenization performed with either 1% formaldehyde in Dulbecco's phosphate buffered saline or 2mM DSG (Proteochem) in Dulbecco's phosphate buffered saline. Nuclei were stained overnight with PU.1-PE (Cell Signaling 81886S), OLIG2-AF488 (Abcam 225099) or SALL1 AF647 (Thermo, clone NRNSTNX 51-9279-82) or NEUN-AF488 (Millipore MAB 377X). Nuclei were washed the following day with 4 mL FACs buffer, passed through a 40 uM strainer, and stained with 0.5 ug/mL DAPI. Nuclei for each cell type were sorted with a Beckman Coulter MoFlo Astrio EQ cell sorter and pelleted at 1600xg for 5 minutes at 4°C in FACs buffer. Nuclei pellets were snap frozen and stored at -80°C prior to library preparation. Chromatin immunoprecipitation was performed as previously described (Heinz et al., 2018). For H3K27ac ChIP, 500,000-1,000,000 fixed sorted cells or nuclei were thawed on ice and resuspended in ice-cold LB3 (10mM Tris-HCl pH 7.5, 100mM NaCl, 1 mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine), 1x protease inhibitor cocktail (Sigma). Chromatin was sheared by sonication. Samples were sonicated in a 96 Place microtube Rack (Covaris cat#500282) using a Covaris E220 for 12 cycles with the following setting: time 60 seconds, duty cycle 5.0, PIP 175, cycles, 200, amplitude 0.0, velocity 0.0, dwell 0.0. Samples were recovered and spun down at max speed, 4°C for 10 minutes. The supernatant was then diluted 1.1-fold with ice-cold 10% Triton X-100. One percent of the lysate was kept as ChIP input. 25 uL of Dynabeads Protein A were added per sample, in addition to 1ug of a specific antibody for H3K27ac (Active Motif 39685). The samples were rotated overnight at 4°C and were washed as follows the next day: 3x with Wash Buffer I (20mM Tris-HCl pH 7.5, 150 mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100) + protease inhibitor cocktail, 3x with Wash Buffer III (10mM Tris-HCl pH 7.5, 250 mM LiCl, 1% Triton X-100, 1mM EDTA, 0.7% Sodium Deoxycholate)+ protease inhibitor cocktail, 2x with TET (0.2% Tween-20/TE) + 1/3 protease inhibitor cocktail, 1x with TE-NaCl (50mM NaCl + TE), and 1x with IDTET (0.2% Tween-20, 10mM Tris pH8, 0,1mM EDTA). Samples were finally resuspended in TT buffer (10mM Tris pH 8 + 0.05% Tween 20) prior to on-bead library preparation. For SALL1, SMAD4, and P300 ChIPs, 500,0000-2million nuclei were thawed on ice and resuspended in ice-cold RLNR1 buffer (20mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.4% sodium deoxycholate, 1% NP-40, 0,1% SDS, 0.5mM DTT) + 1x protease inhibitor cocktail/PMSF. Samples were sonicated in a 96 Place microtube Rack (Covaris cat#500282) using a Covaris E220 for 20 cycles with the following setting: time 60 seconds, duty cycle 5.0, PIP 175, cycles, 200, amplitude 0.0, velocity 0.0, dwell 0.0. Samples were recovered and spun down at max speed, 4°C for 10 minutes. One percent of the lysate was kept as ChIP input. 10 uL of Dynabead Protein A and 10 uL of Dynabead Protein G beads per sample were coupled to either 4 ug of SALL1 antibody (Abcam, ab41974), SMAD4 antibody (1 μg each of Cell Signaling technology 46535 and 38454), or P300 antibody (1 ug each of EMD Millipore RW128  and Diagenode C15200211). Beads/antibody was added to each sample, which were then rotated overnight at 4°C. The samples were washed with the following buffers: 3x RLNR1 + PIC/PMSF/DTT, 6x LWB-RCNR1 (10mM Tris HCl pH 7.5, 1mM EDTA, 0.7% sodium deoxycholate, 1% NP-40, 250mM LiCl)+ PIC/PMSF, 3x TET, 2x IDTET, and then resuspended in TT for on-bead library preparation. Libraries for ChIP and input samples were prepared with NEBNext Ultra II DNA library prep kit (NEB) reagents according to the manufacturer's protocol on the beads suspended in 25 μL TT (10mM Tris/HCl pH7.5, 0.05% Tween-20), with reagent volumes reduced by half. DNA was eluted and crosslinks reversed by adding 4 μl 10% SDS, 4.5 μl 5 M NaCl, 3 μl EDTA, 4 μl EGTA, 1 μl proteinase K (20 mg/ml), 16 μl water, incubating for 1 h at 55°C, then 30 minutes to overnight at 65°C. DNA was purified using 2 μL of SpeedBeads (GE Healthcare), diluted with 20% PEG8000, 1.5M NaCl to final of 12% PEG, eluted with 25 μl TT.  DNA contained in the eluate was then amplified for 12-14 cycles in 25 μl PCR reactions using NEBNext High-Fidelity 2X PCR Master Mix (NEB) and 0.5 mM each of primers Solexa 1GA and Solexa 1GB. Resulting libraries were size selected by gel excision to 200-500 bp, purified, and single-end sequenced using a HiSeq 4000 or paired end on a NovaSeq 6000.