title: GSM3416787: CTCF_1cell_rep28; Mus musculus; OTHER
library_construction_protocol: The CUT&RUN protocol was modified from Skene and Henikoff (eLife 2017), who made their modified protocol available online (http://blocks.fhcrc.org/steveh/papers/CUT&RUN_protocol.htm). Mouse ES cells were counted using a TC-10 cell counter (Biorad) and diluted manually to respective cell amounts. Cells were pelleted at 600g for 3 minutes at 4°C, the supernatant was discard and cells were washed with 1mL cold PBS. Cells were pelleted at 600g for 3 minutes at 4°C, the supernatant was discard and cells were resuspended in 1mL cold nuclear extraction (NE) buffer (20mM HEPES-KOH, pH 7.9, 10mM KCl, 0.5mM Spermidine, 0.1% TritonX-100, 20% glycerol, freshly added protease inhibitors). Cells were pelleted at 600g for 3min at 4°C, the supernatant was discard and cells were resuspended in 600uL NE buffer. During the cell washes, Concanavalin A beads (Polysciences) were prepared. For 500,000 cells 200uL bead slurry were used, for 50,000 cells 150uL bead slurry were used, for 5,000 cells, 100uL bead slurry were used, for 500 cells and 50 cells 50uL bead slurry were used, and for 10 cells 20uL bead slurry were used. Beads were withdrawn and transferred to a microfuge tube containing 3X volume cold Binding buffer (20mM HEPES-KOH, pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2). Beads were washed twice in 1mL cold Binding buffer and resuspended in 300uL binding buffer. Extracted nuclei were added to beads with gentle vortexing and incubated for 10 minutes at room temperature. After nuclei binding, cytosolic fraction was discarded and beads were blocked with 1mL cold Blocking buffer (20mM HEPES, pH 7.5, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA, 2mM EDTA, freshly added protease inhibitors) which was added with repeated gentle pipetting and incubated for 5 minutes at room temperature. Supernatant was discarded and nuclei/beads were washed in 1mL cold Wash Buffer (20mM HEPES, pH 7.5, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA, freshly added protease inhibitors) and resuspended in 250uL cold Wash Buffer. Primary antibody was added with gentle vortexing of the nuclei in 250uL cold Wash Buffer to a final concentration of 1:100. Samples were incubated with rotation at 4°C for 2 hours. Supernatant was discarded and samples were washed twice in 1mL cold Wash Buffer. Supernatant was discarded and samples were resuspended in 250uL cold Wash Buffer. ProteinA-MNase (kindly provided by Steven Henikoff) was added with gentle vortexing of the nuclei in 250uL cold Wash Buffer to a final concentration of 1:400. Samples were incubated with rotation at 4°C for 1 hour. Supernatant was discarded and samples were washed twice in 1mL cold Wash Buffer. Supernatant was discarded and samples were resuspended in 150uL cold Wash Buffer. Samples were equilibrated to 0°C on ice water for 5-10 minutes. 3uL 100mM CalC2 was added during gentle vortexing, samples were flicked quickly to mix and returned to 0C on ice water. After 5 minutes of digestion, reactions were stopped with addition of 150uL 2XSTOP buffer (200mM NaCl, 20mM EDTA, 4mM EGTA, 50ug/mL RNaseA, 40ug/mL glycogen, 10pg/mL yeast spike-in DNA). Samples were incubated at 37°C for 20 minutes to activate RNase and release fragments. Samples were centrifuged at 16,000g for 5minutes and supernatants were transferred to a new microfuge tube while pellets and beads were discarded. Following addition of 3uL 10% SDS and 2.5uL 20m/gmL ProteinaseK, samples were mixed by inversion and incubated at 70°C for 10 minutes. DNA was cleaned using PCI followed by chloroform extraction, and precipitated with glycogen and ethanol. DNA was pelleted with a high speed spin at 4C, washed, air dried for ~5 minutes and resuspended in 36.5uL 0.1XTE. Libraries were prepared using a modification of the Henikoff available protocol. DNA end-repair, phosphorylation, and A-tailing was performed in a single reaction, as follows. T4 DNA Polymerase (NEB) was diluted 1:20. 5uL 10X T4 DNA ligase buffer (NEB), 2.5uL 10mM dNTPs, 1.25uL 10mM ATP, 3.13uL 40% PEG4000, 0.63uL T4 PNK (NEB), 0.5uL diluted T4 DNA Polymerase, and 0.5uL Taq polymerase (homemade) was added to 36.5uL of CUT&RUN enriched DNA. Samples were incubated at 12°C for 15 minutes, 37°C for 15 minutes, followed by 72°C for 20 minutes in a thermocycler. Samples were put on ice immediately and the following adapter ligation reaction was performed. PE inline Illumina adapters were used for these experiments. 55uL of 1X Quick ligase buffer (NEB), 5uL Quick ligase (NEB), and 5uL of 1.5uM adapter mix was added to 50uL of A-tailed DNA and samples were incubated at 20°C for 15 minutes in a thermocycler. Immediately following adapter ligation, samples were cleaned using Ampure XP beads (Beckman Coulter). Beads were warmed to room temperature during adapter ligation and 38uL well-mixed beads were added to libraries. Samples were mixed thoroughly and incubated for 15 minutes at room temperature. Following solution clearing on a magnetic rack, supernatants were discarded and beads were washed two times with 200uL 80% EtOH. Samples were briefly spun, residual liquid was discarded, and beads were allowed to air dry for ~5 minutes. DNA was eluted from beads by resuspending beads in 30uL 10mM Tris-HCL pH 8.0 and incubation at room temperature for 2 minutes. Following solution clearing on a magnetic rack, 27.5uL DNA was transferred to a 0.2mL PCR tube and library enrichment was performed. Libraries were enriched for 14-19 cycles of PCR, with the following conditions. 10uL 5X KAPA buffer, 1.5uL 10mM dNTPs, 5uL 20uM PE PCR 1.0, 20uM PE PCR 2.0, 1uL KAPA hotstart HiFi polymerase (KAPA) was added to 27.5uL of library DNA. The following PCR program was used to enrich the samples: 98°C 45 seconds, 98°C 15 seconds, 60°C 10 seconds, steps two and three were repeated specified number of times, 72°C 1 minute. Following library enrichment, samples were loaded on an agarose gel and DNA corresponding to 150-700 bases was extracted using the Qiagen extraction buffer and Econospin columns. Size distribution of libraries was determined using a Fragment analyzer, and libraries were sequenced on an Illumina NextSeq 500.