GSM9338125: Tada2b_2_H3K9Ac; Mus musculus; ChIP-Seq
CD201+CD150+KSL cells were fixed with 1% formaldehyde (10 minutes incubation at room temperature with rotation), then washed in PBS. Cell pellets were then resuspended in 120 µl of lysis buffer (50mM Tris pH8, 10mM EDTA, 0.5% SDS, 1.2 ul PIC per every 120 µl in ddH2O), and sonicated in Covaris AFA fiber microtubes (Covaris, cat. 520045) aiming for 200 bp fragments. SDS was neutralized with 1% Triton (25 µl of 10% Triton X100 for every 225 µl of sample), and samples were incubated at room temperature for 10 minutes with rotation. 5ul of fresh Protein A dynabeads (ThermoFisher, cat. 10001D, washed with PBS to remove the residual storage buffer), were added per reaction, followed by incubation at 4°C for 30 min with rotation. Samples were then placed on a magnet and cleared chromatin was collected, 5% of each reaction was allocated to a new DNA LoBind tube and stored at -20°C as input control.  Protein A dynabeads (ThermoFisher 10001D, 10 µl per antibody per sample) were washed twice with the wash buffer (200 µL PBS with 0.5% BSA + 1ul PIC per antibody per sample), and resuspended in 200 µl wash buffer per reaction. Beads were then transferred to fresh DNA LoBind tubes and 1 µl of antibody was added to each reaction. The beads were then incubated for 3-4 hours at 4°C with rotation. The beads were then washed twice in a new wash buffer (500 µl PBS + 0.5% FCS +1 µl Tween, 250 µl for each wash), 250ul of cleared chromatin was added to each antibody-bead tube followed by an overnight incubation at 4°C with rotation. The following day each reaction was washed three times with 150 µl of RIPA buffer (50 mM HEPES pH 7.6, 50 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na Deoxycholate in ddH2O), once with 150 µl of TE buffer, and once with 150 µl of 10mM Tris pH 8. The beads were then re-suspended in 25 µl of tagmentation buffer (prepare 5X stock of 50mM Tris pH8, 25mM MgCl2 and 50% dimethylformamide in ddH20, and dilute to 1X with ddH20) pre-warmed to 37°C, followed by addition of 5 µl of diluted Tn5 transposase (Illumina, cat. 20034197, 1ul of Tn5 per 4 µl of tagmentation buffer) and incubation at 37°C for 5 min with rigorous mixing. The beads were then immediately washed once with the RIPA buffer, once with 10mM Tris pH8 (150 µl per tube) and resuspended in ddH2O (22.5 µl per tube). For input controls, 2.5 µl 50 mM MgCl2  was added to 12.5 µl of input chromatin sample, followed by the addition of 6 µl of 5x tagmentation buffer, 1 µl of Tn5 and 8 µl of ddH2O; samples were then incubated at 37°C for 10 min (mixed after 5 min), placed on ice and 20 µl were taken for library preparation step.  For library preparation, the beads were supplemented with 25 µl of 2X NEBNext Ultra II Q5 Mastermix (New England Biolabs, cat. M0544S), 1.25 µl of universal adapter primer and 1.25 µl of index adapter primer and subjected to the following PCR programme: 72°C 5 min, 95°C 5 min, 12 cycles of (98°C 10 sec, 63°C 30 sec, 72°C 3 min), 72°C 5 min, 12°C indefinite hold. Upon library preparation PCR, the tubes were transferred to the magnet and supernatant was put in fresh DNA LoBind tubes. 50 µl of the AMPure XP beads (Beckman Coulter, cat. A63881) were added to each tube, and the samples were incubated at room temperature for 2 minutes. The beads were then washed twice with 150 µl of freshly prepared 80% ethanol, incubated at room temperature for 2 min, ethanol was removed and the beads were air-dried, resuspended in 11 µl ddH2O and incubated at room temperature for 2 min again. The tubes were put on the magnet and 10 µl were collected from each tube. QC and quantification of the samples were performed using D1000 high sensitivity ScreenTape (Agilent, cat. 5067-5584) and KAPA Library Quantification Kit (Roche, cat. KK4824). Library amplification was performed using KAPA HiFi HotStart Library Amp Kit (Roche, cat. KK2612) according to manufacturer's recommendations.