GSM6857980: Normal, IgG; Homo sapiens; OTHER
CUT&RUN for SOX9 and IgG control in Adenoma and Normal organoids was done using CUTANA ChIC/CUT&RUN kit (Epicypher #14-1048) following manufacturerÆs protocol. Briefly, 5???105 cells were captured with Concanavalin A, permeabilized using 0.01% digitonin and incubated with 0.5?g antibody (anti-Sox9 (CST, #82630) and IgG (# 13-0042; Epicypher) in 50?L antibody buffer (20mM HEPES at pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x protease inhibitor cocktails (EDTA-free tablet; Roche), 2 mM EDTA, 0.01% digitonin) overnight. After removing unbound antibody, pAG-MNase (20X) in 50?L cell permeabilization buffer was added to the cells and incubated for 10 min at RT. Then CaCl2 (2mM) was added to activate MNase and incubated at 4??C for 2 hr. The reaction was stopped using 33?L of 2X STOP buffer and E. coli spike-in DNA (0.5ng) was 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 as described previously (Skene et al., 2018) with a few modifications. Briefly, end repair and dA-tailing were conducted on 6?ng of CUT&RUN eluted DNA for 30 min at 20?C followed by 1hr at 50?C. After adaptor ligation for 30 min at 20?C, the DNA fragments were purified by 1.75X 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. After quantitative and qualitative analysis, libraries with different indexes were pooled and sequenced on Illumina Novaseq platform with paired-end 150-bp reads.