GSM4143971: HUtSMC-crAP-1-INPUT-Rep-1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Uterus
Cell type
Myometrium
MeSH Description
The smooth muscle coat of the uterus, which forms the main mass of the organ.
Attributes by original data submitter
Sample
source_name
Human uterine smooth muscle cells (HUtSMCs)
genotype
AP-1 depleted
tissue
Myometrium
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
16% paraformaldehyde was added to full media-containing HUtSM cell plates to a final concentration of 1% paraformaldehyde and incubated at room temperature for 10min and then quenched with 0.125M glycine for 10min at room temperature with rotation. Cross-linked cells were then incubated in ChIP lysis buffer 1 (50mM HEPES-KOH [pH 7.6], 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100, 1X Protease inhibitor cocktail [Roche, cat # 4693132001]) followed by end-over-end rotation at 4°C for 15min and then low speed centrifugation at 4°C. Samples were resuspended in ChIP lysis buffer 2 (10mM Tris-HCl [pH 8.0], 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 1X Protease inhibitor cocktail [Roche, cat # 4693132001]) followed again by end-over-end rotation at 4°C for 15min and sample recovery by low speed centrifugation at 4°C. Samples were then resuspended in ChIP lysis buffer 3 (10mM Tris-HCl [pH 7.5], 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% sarkosyl) and sonicated in an ice water bath (Misonix, setting 6 [~6 W power output], 12 cycles of 15sec on and 45sec off). Triton X-100 was added to a final concentration of 1% and samples were centrifuged at 20,000 x g at 4°C for 20min, with recovery of chromatin-containing supernatant. Solubilized chromatin was incubated overnight with antibody at 4°C with end-over-end rotation. KAPA Hyper Prep kit (Kapa Biosystems, cat # KK8502) was used for end-repair, A-tailing, and adapter ligation with TruSeq index adapters, all according to manufacturer's instructions. A 0.6X-0.8X AMPure XP bead (Beckman Coulter, cat # A63881) size selection was carried out after adapter ligation to enrich for sub-nucleosomal DNA fragments followed by 12 cycles of PCR amplification.