title: GSM9441736: 22Rv1-dTAG-FOXA1 cells (Input); Homo sapiens; ChIP-Seq
library_name: GSM9441736
library_construction_protocol: Cells were washed with PBS, trypsinized, and crosslinked with 1.1 % formaldehyde (final) in 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA for 10 minutes at room temperature. Crosslinking was quenched with 125 mM glycine for 5 minutes. Cells were washed three times with ice-cold PBS and snap-frozen at –80 °C until processing. For chromatin preparation, pellets were resuspended in Lysis Buffer 1 (LB1; 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10 % glycerol, 0.5 % NP-40, 0.25 % Triton X-100, 1× Halt™ Protease Inhibitor Cocktail (Thermo Fisher)) and rotated for 10 minutes at 4 °C, followed by centrifugation at 1,400 × g for 5 minutes at 4 °C. Pellets were then washed with Lysis Buffer 2 (LB2; 10 mM Tris pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease inhibitor) and resuspended in Sonication Buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 0.1 % sodium deoxycholate, 0.5 % SDS, 1× protease inhibitor). Chromatin was sheared using a Diagenode Bioruptor (25 cycles, 30 sec on/off, 10 °C). 50µL of whole cell extract (input) for the DMSO sample was moved to a new tube and stored at -20 °C until the reverse crosslinking step. Sheared chromatin was diluted to 0.1 % SDS and incubated overnight at 4 °C with antibodies conjugated to Dynabeads™ Protein G (Thermo Fisher): 10 µg FOXA1 (Novus Biologicals) or 5 µg H3K27ac (Diagenode). Beads were washed twice with Sonication Buffer (without SDS), once with High-Salt Sonication Buffer (500 mM NaCl, without SDS), once with LiCl Wash Buffer (20 mM Tris pH 7.5, 1 mM EDTA, 250 mM LiCl, 0.5 % NP-40, 0.5 % sodium deoxycholate), and once with TE + 50 mM NaCl. Bound chromatin was eluted in 200µL Elution Buffer (50 mM Tris pH 7.5, 10 mM EDTA, 1 % SDS) at 65 °C for 15 minutes. The input sample was thawed and 150µL of elution buffer was added before crosslinking with the other samples at 65 °C overnight (13 h). Samples were treated sequentially with 8µL 10mg/mL RNase A (37 °C, 2 h), 7µL of 300mM CaCl2 in 10mM Tris at pH 8, and 4µL 20mg/mL Proteinase K (55 °C, 30 min), followed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1). The aqueous phase was isolated and DNA precipitated at -80 °C for 2-3 h. DNA fragments (300-500 bp) were purified and processed for library preparation using the ThruPLEX® DNA-seq Kit (Takara Bio), indexed with DNA Unique Dual Index Kit -24U Set A (Takara), cleaned with AMPure XP beads (Beckman Coulter), and sequenced on an Illumina NextSeq 2000 (single-end, 75 bp).
identifiers.namespace: GEO
identifiers.id: GSM9441736_r1