GSM8785595: Rloop (S9.6) CUT&Tag ERCC1 WT mESC 2; Mus musculus; OTHER
Quantitative CUT&Tag experiments were performed as described previously (17) with modifications. Briefly, 2 x 105 mouse embryonic stem cells were harvested, washed once with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine), and then incubated in 200 mL of antibody buffer (wash buffer with 1% BSA, 2 mM EDTA, 0.01% NP-40 and 0.05% digitonin) with 1:100 purified FLAG-tagged BG4 antibody or S9.6 (Sigma-Aldrich) antibody dilution at 4°C by rotating overnight. The next day, the primary antibody was removed by centrifugation at 600 xg for 4 minutes at 4ºC, and cells were washed with 200 mL of dig-wash buffer (wash buffer with 1% BSA, 0.01% NP-40 and 0.05% digitonin) three times. After washing, BG4 antibody-incubated cells were resuspended in 200 mL of dig-wash buffer with 1:100 dilution of mouse anti-FLAG antibody (Sigma-Aldrich) and incubated at room temperature for 1 hour with slow rotation. Cells were washed with 200 mL 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 rabbit anti-mouse antibody (Sigma-Aldrich) in 200 mL of dig-wash buffer at room temperature for 1 hour with slow rotation. After a brief wash with dig-wash buffer as above, cells were resuspended in 200 mL of dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.05% digitonin, 0.01% NP-40, 0.5 mM spermidine, 1% BSA) with 1:100 dilution of assembled pA-Tn5 and incubated at room temperature for 1 hour with slow rotation. pA-Tn5-bound cells were washed with 1 mL of dig-300 buffer three times, followed by tagmentation in 200 mL of tagmentation buffer (dig-300 buffer with 10 mM MgCl2) at 37°C for 1 hour with shaking at 800 rpm. After tagmentation, 15 mM EDTA, 500 mg/mL proteinase K and 0.1% SDS were added and further incubated at 63°C for another 1 hour to stop tagmentation and digest protein. Genomic DNA was extracted and purified with DNA Clean & Concentrator-5 (Zymo research). A spike-in was used where indicated to provide an external scaling factor for quantitative comparison across conditions. Briefly, G4 CUT&Tag was performed on S2 cells in parallel with the mouse samples. After tagmentation and genomic DNA purification, the tagmented DNA was quantified using Qubit assay. The tagmented samples were normalized to the same DNA concentration, and a constant amount (5% of sample) of tagmented Drosophila DNA ('spike-in') was added to each sample, at a ratio of 1:20. Afterwards, library PCR was performed as described above. An alternative method, mixing 5% S2 cells into the harvested cells before CUT&Tag procedure was also evaluated but showed larger variation due to the error attached to cell counting. To generate G4 or R-loop libraries, purified genomic DNA was amplified with the universal i5 primer and barcoded i7 primer using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs). The library PCR products were cleaned up with Agencourt AMPure XP beads (Beckman Coulter) and sequenced on an Illumina Nextseq 2000 instrument (illumina).