title: GSM5259796: mES R-loop CUT&Tag Rep1 Batch1; Mus musculus; OTHER
library_construction_protocol: CUT&Tag experiment was performed as described previously (Kaya-Okur et al. 2019) with minor modifications" Briefly, 1x10e5 cells were harvested, washed with wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine), and immobilized to concanavalin A膨oated beads (Bangs Laboratories, BP531) with incubation at room temperature for 10min. The beads-bound cells were incubated in 200?l of primary antibody buffer (wash buffer with 2mM EDTA and 0.05% digitonin) with 1:100 BG4 antibody or S9.6 (Millipore, MABE1095) antibody dilution at 4? by rotating overnight. The next day, the primary antibody buffer was removed and cells were washed with 800?l of dig-wash buffer (wash buffer with 0.05% digitonin) three times. After washing, BG4 antibody-incubated cells were resuspended with 200ul of dig-wash buffer with 1:100 dilution of anti-FLAG antibody (Sigma, F1804) and incubated at room temperature for 1 hour by rotating. Cells were washed with 800?l of dig-wash buffer briefly three times to remove unbound antibodies. S9.6-treated or anti-FLAG treated cells were incubated with 1:100 dilution of anti-mouse antibody (Sigma, M7023) in 200?l of dig-wash buffer at room temperature for 1 hour by rotating.After brief wash with dig-wash buffer, cells were resuspended in 200ul of dig-300 buffer (20mM HEPES pH 7.5, 300mM NaCl and 0.5mM spermidine, 0.01% digitonin) with 1:200 dilution of pA-Tn5 adapter complex and incubated at room temperature for 1 hour by rotating. pA-Tn5-bound cells were washed with 800ul of dig-300 buffer three times, followed by tagmentation in 200ul of tagmentation buffer (dig-300 buffer with 10mM MgCl2) at 37? for 1 hour. After tagmentation, 15mM  EDTA, 500?g/ml proteinase K and 0.1% SDS were added and further incubated at 63? for another 1 hour to stop tagmentation and digest protein. Genomic DNA was extracted and purified with DNA Clean & Concentrator-5 (Zymo research, D4013). To generate G4 or R-loop libraries, purified genomic DNA were mixed with the universal i5 primer and barcoded i7 primer, and amplified with NEBNext Ultra II Q5 Master Mix (NEB, M0544). The library PCR products were cleaned up with Agencourt AMPure XP beads (Beckman Coulter, A63881) and sequenced with Illumina Nextseq 500 instrument.