title: GSM4002919: CUTRUN1_Parental_flag; Homo sapiens; OTHER
library_construction_protocol: Cut & Run was performed according to the protocol described in Meers et al.(2019) with slight modifications. All reactions were performed at 4ºC unless stated otherwise. Each centrifugation step was performed at 600 g for 3 min. 4 x 106 cells was used for each reaction. Cells were washed twice with PBS and resuspended in Buffer NE1 (20 mM HEPES-KOH pH7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1% Triton X-100, 20% Glycerol, Proteinase Inhibitor). After 10 min, cells were collected and incubated in Buffer 1 (20 mM HEPES-KOH pH7.9, 150 mM NaCl, 2 mM EDTA, 0.5 mM Spermidine, 0.1% BSA, Proteinase Inhibitor) for 5 min. Cells were collected, washed once with Buffer 2 (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, Proteinase Inhibitor) and resuspended in CUT&RUN Buffer 2. Samples were incubated with antibodies for 2 hours or overnight. Samples containing primary mouse antibodies were washed twice with Buffer 2 before incubation with anti-mouse rb IgG (Jackson ImmunoResearch, 315-005-003) for one hour. Nuclei was then collected, washed twice with Buffer 2 and incubated with pA-MNase fusion protein (150 ng) for one hour. Nuclei was washed once with Buffer 2 and once with low salt buffer (3.5 mM HEPES, Proteinase Inhibitor). Upon resuspension in low salt buffer, 10 mM CaCl2 was added to each reaction. MNase digestion was stopped after 30 min with the addition of Buffer 2 (supplemented with 10 mM EDTA, 20 mM EGTA and 28.8 pg yeast spike in). Samples were incubated for 15 min at 37 ºC. DNA was purified using NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL). CUT&RUN libraries were prepared using Ovation Ultralow System V2 (NuGEN, 0344NB). CUT&RUN libraries were sequenced on a HiSeq X platform (Illumina) using paired end sequencing. ATAC-seq was performed according to the protocol in Buenrostro et al. (2015). 50,000 cells were suspended in cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40). Crude nuclei were isolated by centrifugation at 500 g for 10 min and resuspended in 25 ml of TD Buffer (2x reaction buffer, Illumina). Nuclei pellet was collected (500 g, 5 min) and suspended in transposition reaction mix (25 ml, 2.5 ml TDE1, 22.5 ml l H2O). The reactions were incubated at 37ºC for 30 min with gentle mixing. DNA was immediately purified using Qiagen MinElute PCR Purification kit. ATAC-seq libraries were PCR amplified according to protocol and purified using Qiagen MinElute PCR Purification kit. Double sided bead purification (0.5x beads volume, followed by 1.3x beads volume) was performed using AMPure XP beads to enrich for fragments between 150 bp to 1000 bp. Library quality and fragment sizes were accessed using Bioanalyzer (Agilent). ATAC-seq libraries were sequenced with 150 bp paired end sequencing on HiSeq X (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4002919