title: GSM8428065: BT16 cells, H3K27me3, TM, replicate 1, CUT&RUN; Homo sapiens; OTHER
library_construction_protocol: CUTANA protocol (ChIC/CUT&RUN kit Version 3) was used for CUT&RUN assay. Cleavage under target and release using nuclease (CUT&RUN) 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. BT16 ATRT cells were collected using 0.05% trypsin, 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 600 g for 3 min, for a total of two washes. Cells were resuspended in 100 ul/sample RT Wash Buffer, and then an aliquot of 100 ul washed cells was added 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 cleared and the supernatant was removed. 50 ul of cold Antibody Buffer was added to each sample and mixed by pipetting; 1ul of antibody IgG (Rabbit IgG Negative Control, EpiCypher, 13-0042 K), H3K4me3 (Positive Control, EpiCypher, 13-0041), H3K27Ac (Cell Signaling, #8173), H3K27me3 (Cell Signaling, #9733), was added to each sample, mixed and incubated on a nutator overnight at 4 °C. The tube was placed on a magnet until slurry cleared, 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 pipetted to remove supernatant, for a total of 2 washes. 50 ul of cold Digitonin Buffer was added to each sample and mixed. 2.5ul of CUTANA pAG-MNase (20Xstock) was added to each sample, mixed, and incubated for 10 min at RT. Tube was placed on a magnet and supernatant was removed. The beads were washed 2 times with cold Digitonin Buffer. Supernatant was removed, 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 and mixed, and then incubated on nutator 2 hrs at 4 °C. 33 ul/sample of Stop buffer 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 37 °C in a thermocycler. Tubes were placed on a magnet stand until slurry cleared, and the supernatant was transferred to a 1.5 ml tube. Finally, the DNA was purified using CUTANA DNA Purification Kit (EpiCypher, 14-0050) according to the manufacturer instructions, and 1 ul of DNA was used for quantification by Qubit. 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&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 75 °C and run the program 30 minutes at 20°C, 60 minutes at 50 °C and hold at 4 °C. 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 20 °C for 15 min with heated lid off. 3 ul of USER Enzyme was added to the reaction and incubated at 37 °C for 15 min with heated led set to 47 °C. 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: 98 °C for 45s, 98 °C 15s and 60 °C 10s for 16 cycles, 72 °C for 1 min and hold at 4 °C. 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.