rabbit-monoclonal VDR antibody (Cell Signalling, custom made 2 mg/mL, MA, USA)
treatment
EtOH
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
DNA-protein crosslinking was done by incubation with 1% formaldehyde (Thermo Fisher Scientific) for 8 min in rotation at 4ºC. Crosslinking was quenched with 1.25 mM glycine (Bio-Rad) in PBS for 5 min. We transferred the organoid solution to a 50 mL conical tube for centrifugation at 2500 rpm for 5 min at 4ºC. Supernatant was removed and the pellet was washed twice in cold PBS, transferred to a 1.5 mL eppendorf tube and lysed in 1 mL ChIP-lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1 and 1x protease cocktail inhibitors (cOmplete, EDTA-free (Roche, Basel, Switzerland)) for 1 h. We used a 21G syringe to homogenize the solution and force the lysis. Samples were sonicated for 20 min at maximum intensity at 4ºC in a S220 Focused-ultrasonicator (Covaris, MA, USA). Both ChIP'ed and input DNA samples were processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters with "NEBNext Ultra II DNA Library Prep Kit for Illumina" (NEB, MA, USA). Adapter-ligated libraries were completed by limited-cycle PCR and extracted with a single double-sided SPRI size selection. Cluster generation was performed on an Illumina flow cell and sequenced in the 50 base single-read format on an Illumina HiSeq2500 instrument by following manufacturer's protocols.