GSM8267137: D458 cells, H3K4me1, replicate 2, ChIP; Homo sapiens; OTHER
Beads were prepared using 10 ul/sample of CUTANA Concavalin A Conjugated Paramagnetic Beads (EpiCypher, SKU:21-1401). Beads were transferred to a 1.5 ml tube and placed in a magnetic separation rack. Supernatant was removed and beads were washed 2 times with 100 ul/sample of cold Bead Activation Buffer (20 mM HEPES pH7.9, 10 mM Kcl, 1 mM CaCl2, 1 mM MnCl2). Beads were then resuspended in 10 ul/sample of cold Bead Activation Buffer and aliquot 10 ul/sample of activated bead slurry into 8-strip tube and kept on ice.  Cells were collected using 0.05% trypsin and washed with 100 ul/sample of Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete EDTA-free Protease Inhibitor (1187358001)) at room temperature and centrifuged at RT, 600 g for 3min, for a total of two washes. Cells were resuspended in 100 ul/sample RT Wash Buffer and then aliquot of 100ul washed cells to each 8-strip tube containing 10 ul of activated beads and mixed by pipetting. Cells were incubated with bead slurry for 10 min at RT. Tube strip was placed on a magnetic separation rack until slurry clears and the supernatant was removed. 50 ul of cold Antibody Buffer was added to each sample and mixed by pipetting; 1 ul of antibody, IgG (Rabbit IgG Negative Control, EpiCypher, 13-0042K), PolI (Rpb1 CTD (4H8), Cell Signaling, #2629S), pPolI (pRpb1 CTD (S2), Cell Signaling, #13499S), Brd4 (Brd4, EpiCypher, 13-2003), ENL (MLLT1/ENL (D9M4B);Cell Signaling, #14893) was added to each sample, mixed and incubated on a nutator overnight at 4oC. Tube was placed on a magnet until slurry clears and the supernatant was removed. While beads were on magnet, 250 ul of cold Digitonin Buffer was added directly onto beads of each sample and then pipet to remove supernatant, for a total of 2-washes. 50 ul of cold Digitonin Buffer was added to each sample and mixed. 2.5 ul of CUTANA pAG-MNase (20Xstock) was added to each sample, mixed and incubated for 10min at RT. Tube was placed on a magnet and supernatant was removed. Beads were washed 2 times with cold Digitonin Buffer. Supernatant and 50 ul of cold Digitonin Buffer was added to each sample and mixed. Tubes were placed on ice and 1 ul of 100 mM CaCl2 was added to each sample, mixed, and then incubated on nutator 2 hrs at 4oC. 33 ul/sample of Stop buffer, to stop the MNase activity by chelating Ca++ ions, containing 0.5 ng/sample of E.coli Spike-in DNA, was added to the samples, mixed by pipetting  and incubated for 10 min at 37oC in a thermocycler. Tubes were placed on a magnet stand until slurry cleared and the supernatant was transferred to a 1.5ml tube. Finally, the DNA was purified using CUTANA DNA Purification Kit (EpiCypher, 14-0050) according to the manufactory instructions and 1ul of DNA was used to be quantified using Qubit and library preparation. The NEBNext Ultra DNA Library Prep Kit for Illumina (NEB #E7645S) with Dual Index Primers (NEB #E7600) were used to library preparation for ChiPseq and Cut and Run samples sequencing.   For the NEBNext end prep, 1x TE was added to the DNA to get final volume of 50 ul and mixed with 3ul of NEBNext Ultra II End Prep Enzyme Mix and 7ul of NEBNext Ultra II End Prep Reaction Buffer, placed in thermocycler with heated lid set to 75oC and run the program 30 minutes at 20oC, 60 minutes at 50oC and hold at 4oC. The adaptor ligation reaction was performed with 60u l of Prep Reaction Mixture, 2.5ul of NEBNext Adaptor for Illumina diluted following the manufacture recommendations, 30 ul of NEBNext Ultra Ligation Master Mix and 1ul of NEBNext Ligation Enhancer, incubated at 20oC for 15 min with heated lid off. 3 ul of USER Enzyme was added to the reaction and incubated at 37oC for 15 min with heated led set to 47oC. For the cleanup of the adaptor-ligated DNA without size selection, 1.1x of AMPure XP Beads were added to the Adaptor Ligation reaction and samples were incubated for 5min at room temperature. Tubes were placed on a magnetic stand to separate beads from supernatant, after 5 min, supernatant was removed, and beads were washed 2 times with 200ul of 80% freshy prepared ethanol. Beads were air dried for up 5 min while tubes were on magnet with lid open. DNA was eluted from the beads by adding 17 ul of 0.1XTE buffer.  For the PCR enrichment of adaptor-ligated DNA step, to 15 ul of the Adaptor Ligated DNA fragments was added 25ul of NEBNext Ultra Q5 Master Mix, 5 ul of Index primer i7 and 5ul Universal PCR Primer i5, and PCR amplification was performed following the cycling conditions: 98oC for 45s, 98oC 15s and 60oC 10s for 16 cycles, 72oC for 1 min and hold at 4oC. Followed by a Clean Up of the PCR reaction using AMPure XP Beads.  The DNA was quantified using Qubit and the quality and size of libraries were checked with the Bioanalyzer and sent for sequencing at the Genomics Shared Resource (sequencing core) of CU Anschutz Medical Campus.