title: GSM8422219: SCAF1AID_CUTnRUNseq_HA_SCAF1_Auxin_Rep3; Homo sapiens; OTHER
library_construction_protocol: Cells were detached, and washed in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine). After washing, cells were coupled to ConA-coated magnetic beads (Polysciences Europe) and subsequently permeabilized and followed by addition of primary antibody in antibody binding buffer (wash buffer, 0.05% digitonin, 2 mM EDTA) over night at 4 °C. Cells were wased in Dig-Wash buffer (Wash buffer, 0.05% digitonin) and MNase (700ng/ml) was added to each sample for 1 hr at 4 °C. After washing with Dig-wash buffer and Low-salt Rinse buffer (20 mM HEPES pH 7.5, 0.5 mM Spermidine, 0.05% digitonin) incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.05% digitoin) was added for 30 min at 4°C. STOP buffer (170 mM NaCL, 20 mM EGTA, 0.05% digitonin, 20 ug/ml RNAse A, 25 ug/ml Glycogen was added to stop the MNase digestion. DNA fragments were released at 37 °C for 30 min and proteins digested for 1 h at 50 °C after adding 0.01 % SDS and 10 mg/ml Proteinase K followed by Phenol Chloroform extraction. Precipitated DNA was resuspended in 30 ul 0.1x TE buffer Libraries were prepared with NEBnext Ultra II DNA Library Prep Kit (New England Biolabs). Libraries were PCR amplified for 16-18 cycles. The concentration and size distribution of the libraries were determined on a Fragment Analyzer using the NGS Fragment High Sensitivity Analysis Kit (1-6,000 bp; Agilent Technologies). The libraries were sequenced on the NextSeq2000 Illumina platform. Base calling was performed using Illumina's BaseSpace platform.