GSM7574978: TotalRNAPol_WT-2i_DMSO_rep.1; Mus musculus; OTHER
Samples were extracted using CUTANA ChIC/CUT&RUN kit (Epicypher #14-1048) following manufacturer's protocol. Briefly, 5 × 105 cells were captured with Concanavalin A, permeabilized using 0.01% digitonin and incubated with 0.5 μg antibody in 50 μL antibody buffer (20 mM HEPES at pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x protease inhibitor cocktails [EDTA-free tablet; Roche], 2 mM EDTA and 0.01% digitonin) for overnight. After removing unbound antibody, micrococcal nuclease fused to proteins A and G (pAG-MNase (20X) diluted in 50 μL cell permeabilization buffer was added to the cells and incubated for 10 min at RT. CaCl2 (2 mM) was added to activate MNase and incubated at 4 °C for 2 hr. The reaction was stopped using 33 μL of 2X STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A, 50 µg/mL glycogen) and E. coli spike-in DNA (0.5ng) added as a control. The DNA from the released chromatin in the supernatant was purified and then quantified using Qubit dsDNA HS assay kit (Agilent Technologies). CUT&RUN libraries were constructed using NEBNext Ultra II DNA library preparation kit. Briefly, end repair and dA-tailing were conducted on 6 ng of CUT&RUN eluted DNA for 30 min at 20°C followed by 30 min at 65°C. After adaptor ligation for 30 min at 20°C, the DNA fragments were purified by 1X vol of AMPure XP beads (Beckman Coulter) followed by 10-12 cycles of PCR amplification with Next Ultra II Q5 master mix. The PCR products were purified with 1X vol of AMPure XP beads. Library quality was determined by checking the fragment profile using Agilent Tape Station and concentration of the libraries estimated using Qubit dsDNA high sensitivity kit as well as KAPA Library Quantification Kit. Libraries with different indexes were pooled and sequenced on Illumina NovaSeq 6000 platform with 50bp paired end reads.