GSM4752819: UTX ChIPseq mnase pSIN WTUTX 1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
KDM6A
Cell type
Cell type Class
Others
Cell type
UM-UC-1
Primary Tissue
Urinary Bladder
Tissue Diagnosis
Carcinoma Transitional Cell
Attributes by original data submitter
Sample
source_name
UMUC1 bladder cancer cells
cell line
UMUC1
cell type
bladder cancer
genotype
stably expressing pSIN-puro wild type UTX
chip antibody
UTX
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Three days after plating, UMUC1 cells were harvested from 15cm tissue culture plates at ~75% confluency. For each UTX or MLL4 ChIP, ~3x10e7 cells were harvested which yielded ~75 µg of chromatin DNA for immunoprecipitation after going through this protocol. The cells were then fixed as described for the histone modification ChIPs described above. The fixed cells for each sample were resuspended in 20 mL of PBS with 0.5% NP-40 and 1x protease inhibitors, then incubated on ice for 10 minutes and pelleted. Each pellet of fixed cells was divided into two tubes, so there were ~1.5x10e7 cells per tube, and these cell pellets were snap frozen. After thawing the cells, each tube of cells was then resuspended in 600 µL of room temperature micrococcal nuclease (MNase) reaction buffer (50 mM HEPES pH 7.5, 2 mM CaCl2, 0.2% NP-40). Before fragmenting the ChIP samples, the specific lot of MNase (Worthington, LS004798) was tested and a specific amount of MNase (~3-5uL) was determined in order to obtain fragments primarily 300-900 base pairs in length. The 600 µL of resuspended cells were then divided into three tubes of 200 µL and each tube of cells was treated with MNase for 3 minutes at 37°C. The reactions were stopped by adding 20 µL of 0.5M EDTA pH 8.0 and putting the samples on ice. The cells were centrifuged at 13,000xG for 1 minute and the supernatant was removed. The two tubes of cells for each sample were combined and these cells were resuspended in 200uL SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1x protease inhibitors). Then 800 µL of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, 1x protease inhibitors) was added to each sample, which was then sonicated for 5 minutes using the Bioruptor sonicator. The sample was centrifuged at 20,000xG for 10 minutes at 4°C and the supernatant was transferred to a new tube. Then 20 µL was removed to measure the amount of DNA as described above, and the rest was snap frozen and stored at -80°C. After quantifying the DNA concentration for each sample, 75 µg of chromatin was diluted into 2 mL total of ChIP dilution buffer, and 5% (100 µL) of each sample was set aside as an input sample. Then 75 µL of protein A Dynabeads that had been washed 3X with PBS + 0.01% Tween-20 and pre-bound with 20 µL UTX (Cell Signaling, 33510) or MLL4 (Sigma, HPA035977) antibody were added to each chromatin lysate and this mixture was incubated rotating overnight at 4°C. The beads were then sequentially washed on a magnet two times each with low salt buffer, high salt buffer, LiCl buffer and once with TE buffer (see histone modification ChIP section for buffer compositions). After the TE wash the beads were spun down (not separated on the magnet), and resuspended in 200 µL elution buffer (1% SDS, 0.1 M NaHCO3). 100 µL elution buffer was also added to each 100 µL input sample. Samples were incubated at 65°C for 30 minutes while shaking at 800 rpm. Then 8 µL of 5M NaCl was added to each sample before reverse crosslinking overnight at 65°C. The eluate was then treated with RNaseA for 1 hr at 37°C then with Proteinase K for 1 hr at 55°C and DNA was recovered using the Qiagen PCR purification kit. Libraries were prepared using the NEBNext Ultra II DNA Library Prep kit, and then sequenced on a NextSeq500 sequencer. NEBNext Ultra II DNA