title: GSM5311597: BAF180_DOX_BT16; Homo sapiens; ChIP-Seq
library_construction_protocol: Standard one-step crosslinking was performed when targeting histone post-translational modifications (H3K4me3, H3K27ac, H3K4me1 and H3K27me3) for ChIP-sequencing. G401 and BT16 HA-SMARCB1 cells were cultured in T75 flasks (Corning) and treated with 0.01 ng/uL doxycycline or DMSO (mock-treatment) for 72 hours. The cell monolayer or homogenised kidney tissue was washed twice with pre-warmed PBS. Cells were incubated at room temperature in 1% formaldehyde (FA) solution (Millipore Sigma) for 10 minutes before adding 0.125 M glycine (Millipore Sigma) to quench the crosslinking reaction. Two-step crosslinking [67] was performed to target SWI/SNF subunits; BAF170, BAF155, BRG, SS18, ARID1A, BRD9, BAF180, ARID2, BRG7 and HA-SMARCB1 for ChIP-sequencing. 2 mM disuccinimidyl glutarate (DSG; ThermoFisher scientific) was added to cells for 30 minutes at room temperature. Excess DSG was removed by washing twice in PBS and cells were crosslinked with FA solution for 10 minutes as described above. Crosslinked cells were washed twice in PBS and lysed in 5 mL SDS lysis buffer (100 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, 0.02% NaN3, and 1% SDS) containing protease inhibitors (1 μg/mL leupeptin, 1 μg/mL aprotenin, 10 μM PMSF; Millipore Sigma). Cells were snap-frozen in liquid nitrogen and stored at -80oC for later use. ChIP-DNA was quantified using the Qubit® 2.0 Fluorometer (Thermo Fisher Scientific). ChIP-libraries were prepared from 10 ng DNA using the NEBNext®Ultra-library preparation kit for Illumina (New England BioLabs) with Dual indexing NEBNext® Multiplex Oligos (Dual Index Primers Set 1) following manufactures protocol. DNA libraries were purified and size selected (200 – 500 base pairs) by two rounds of AMPure bead purification. Library fragment length was validated using the High Sensitivity (HS) ScreenTape for 2200 TapeStation (Agilent). ChIP library-amplification was validated by qPCR at a set of known positive and negative control genes.