GSM5389611: chip wt es H3K4me2 1; Mus musculus; ChIP-Seq
ChIP Antibodies (H3K4me1 abcam ab8895, H3K27Ac abcam ab4729, H3K36me abcam ab9050, 2 µg antibody per 2x107 cells) were incubated with 25 µL protein A Dynabeads (Thermo Scientific) previously washed twice with blocking buffer (0.5% BSA, 0.5% Tween-20 in PBS) for 2 h at 4 °C in blocking buffer. The conjugated beads were washed twice with blocking buffer before adding the lysate. Lysates were prepared as follows: Cells were trypsinized, washed with PBS, and resuspended with 500 μL PBS per 2x107 cells. The same volume of lysis buffer with 100 U/mL MNase was added, and the cells were incubated for 10 min on ice and then for 15 min at 37 °C. Reactions were stopped by adding 20 mM EGTA followed by centrifugation (20,000 g, 2 min). Supernatants were added to the conjugated beads, and samples were incubated overnight with rotation. Supernatants were removed, and the beads were washed twice with RIPA buffer (10 mM Tris-HCl [pH 8], 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA), twice with RIPA buffer high salt (10 mM Tris-HCl [pH 8], 360 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA), twice with LiCl wash buffer (10 mM Tris-HCl [pH 8], 250 mM LiCl, 0.5% sodium deoxycholate, 1 mM EDTA, 0.5% IGEPAL CA-630 [Sigma]), and twice with 10 mM Tris-HCl [pH 8]. The beads were resuspended in 45 μL 10 mM Tris-HCl [pH 8] and 20 µg RNase A. The beads were incubated for 30 min at 37 °C, and 60 µg proteinase K was added. The beads were further incubated for 2 h at 37 °C. Proteinase K was inactivated by heating to 65 °C for 15 min, and 50 μL elution buffer (10 mM Tris-HCl [pH 8], 300 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) was added, and the samples were incubated in 65 °C for 15 min. DNA was purified from supernatants using Ampure XP beads (Beckman Coulter). DNA was eluted with 22 μL 10 mM Tris-HCl [pH 8].  DNA samples were converted to blunt-ended, phosphorylated DNA using the End-It DNA End-Repair Kit (Lucigen). DNA was purified using ratio of 1:1.8 sample to AMPure XP beads. Adenosine was added to the 3' end of the DNA fragments using Klenow (3'-5' exo-) (New England Biolabs). DNA was purified using ratio of 1:1.8 sample to AMPure XP beads. Ilumina adapters were added by ligation using DNA ligase (New England Biolabs). DNA was purified using ratio of 1:1.2 sample to AMPure XP beads. DNA samples were amplified using KAPA HiFi HotStart (Roche) with 25 μM PE1 and PE2 full-length primers containing Illumina library indices for multiplexing (Table 1). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads and eluted in 22 μL 10 mM Tris-HCl [pH 8]. Aliquots of 15 μL of resulting libraries were run in a 2% agarose gel, and the desired 200-600 bp DNA library fragments were selected and isolated using the PureLink DNA gel extraction kit (Invitrogen). Library quality was confirmed using the Agilent 2200 TapeStation nucleic acids system and the Agilent High Sensitivity D1000 DS DNA kit. The resulting libraries had an average size of 350-550 bp. Size-selected libraries were diluted to 4 nM concentration and combined for paired-end, single index sequencing on the Illumina NextSeq 550 instrument using an illumina 550 High Output v2 (75 cycles) kit. Cycle distribution was 45 cycles for Read 1, 35 cycles for Read 2, and 8 cycles for the library index read.