title: GSM3445667: SMARCA4-infected H1703 SMARCA4 ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were fixed cells with PBS containing 0.3% formaldehyde for 30 mins rocking at 4 C and quenched by addition of 1/10 volume of 2M glycine with 5 min incubation at room temperature. Fixed cells were then washed once with 1x PBS and resuspended in 1 mL swelling buffer (25mM HEPES-NaOH pH7.5, 10mM KCl, 0.1% NP-40, 1.5mM MgCl2). After incubating 10 mins on ice, dounce homogenizer B was used to break cytoplasmic membrane, 20 strokes. 9mL of MNase digest buffer (15mM HEPES-NaOH pH7.9, 15mM NaCl, 60mM KCl, 0.32M sucrose) was added before spinning down at 2000 RPM for 7 mins at 4 C. Pellet(nuclei) was resuspended in 1 mL MNase digest buffer with 3.3 uL of 1 M CaCl2 and incubated for 15 mins at 37C. 0.5 uL of 2000 U/uL MNase per 107 cells was added for another 15 mins incubation for at 37C. Reaction was then quenched with 10uM EGTA before adding 1mL lysis buffer (20mM Tris-HCl, pH 8.0, 200mM NaCl, 2mM EDTA, 1mM EGTA, 0.2% Na-Deoxycholate, 1% N-lauroylsarcosine) and 1% Triton X-100. Lysate was passed through 21G needle 5 times and spinned down at 13000 g at 4C for 15 minutes. 30ug of SMARCA4 antibody (abcam, ab110641) was added to supernatant with overnight end-over-end mixing at 4C. 50 uL of Protein-G Dynabeads (Invitrogene) per IP was added to lysate and incubated at 4C with rotation for at least 2 hours. Beads were washed 4 times with RIPA buffer for 5 minutes each and once with TE, 50 mM NaCl solution. Elution buffer with 100 uL of 100 mM NaHCO3/1% SDS (make fresh) was added to beads and incubated for 17 mins at 65C on Thermos mixer shaking at 1500 RPM. Supernatant was recovered into a new eppendorf tube followed by adding 5 uL of 5 M NaCl. Samples were decrosslinked overnight at 65C. After incubations with RNAseA (3ul, 10ug/mL) for 30 mins at 37C and Proteinase K (5uL, 10ug/mL) for 1 hour at 55C, DNA was cleaned with DNA clean and concentrate kit (Zymo Research). ChIP-seq libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs).
attributes.tag: GEO Accession
attributes.value: GSM3445667