title: GATA6Het_Day3_IgG.bam
organism: Homo sapiens
description: Protocols: hPSCs were routinely cultured in feeder-free conditions on vitronectin-coated (Stemcell Technologies #07180) sterile plates (Corning, Costar) with Essential 8 (E8) media (Life Technologies #A1517001). Subculture was performed every 4-6 days by aspirating the cell culture media, washing the cells once with D-PBS, incubating them in 0.5 mM EDTA (Invitrogen, #15575020) for 5 min at room temperature, aspirating the EDTA and adding appropriate volumes of fresh E8 media (Table 2) to the cells. hPSCs were further physically detached from the plates by gentle pipetting, collected in a 15 ml Falcon tube (BD Falcon) and allowed to settle for 10 min. The supernatant was aspirated and hPSCs were re-suspended in fresh E8 media, and then dissociated into smaller clumps by gentle pipetting. The cells were split at a split ratio of 1:6 and plated onto fresh vitronectin-coated plates containing appropriate volumes of media. Cells were maintained by refreshing E8 media on a daily basis. Co-binding of DNA to DNA-binding proteins was determined by ChIP against GATA6 (Cell Signaling, #5851) on approximately 1 x 107 cells per antibody or control sample. hPSCs grown on 10 cm dishes were differentiated and harvested at the endoderm (D3) stage. 1 x 10 cm dish of cells harvested at D3 was used for one antibody immunoprecipitation (IP). Cells were cross-linked by adding 312.5 µl of 16% formaldehyde (ThermoFisher UK, #11586711) to 5 ml of media to make a final concentration of 1%. The cells were incubated rocking for 10 min at room temperature to allow cross-linking of protein-DNA complexes. The reaction was quenched by adding 312.5 µl of 2 M glycine (Millipore, #357002) to make a final concentration of 0.125 M and incubated for 5 min with rocking. Thereafter, the media was aspirated and cells were washed twice with 5 ml of ice-cold PBS. The cells were detached by scraping into 3 ml of ice-cold PBS containing freshly-added protease inhibitors (10 µl/ml of 5 mg/ml phenylmethylsulfonylfluoride (PMSF; Sigma-Aldrich, #93482), 10 µl/ml of 1 M Sodium Butyrate (Sigma-Aldrich, #303410) and 1 µl/ml of 1 mg/ml Leupeptin (Roche, #11017101001)) and pooled into 15 ml tubes, each tube containing approximately 2 x 107 cells, or 2 IPs. The cells were then centrifuged for 5 min at 1,200 rpm at 4°C to pellet. For all subsequent steps, the samples were kept on ice. For all subsequent buffers used, the aforementioned protease inhibitors were added freshly to the buffers before use. The pelleted cells were subsequently re-suspended in 2 ml of ice-cold Cell Lysis Buffer (10 mM Tris-Cl pH 8.0, 10 mM NaCl and 0.2% NP-40) per 15 ml tube and incubated on ice for 10 min. The cells were then centrifuged for 5 min at 1,800 rpm at 4°C. The supernatant was discarded and the pellet was gently re-suspended in 1.25 ml of ice-cold Nuclear Lysis Buffer (50 mM Tris-Cl pH 8.0, 10 mM EDTA and 1% SDS) per 15 ml tube and incubated on ice for 10 min. 0.75 ml of ice-cold IP Dilution Buffer (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.01% SDS, 1% Triton X-100) was then added per 15 ml tube. The chromatin was then transferred into 15 ml Diagenode sonication tubes containing sonication beads (Diagenode, #C01020031) that were pre-washed twice with 10 ml D-PBS each time and once with 10 ml IP Dilution Buffer. Next, chromatin was sonicated in Diagenode Biorupter Pico for 10 cycles of 30s on/45s off. The sonicated chromatin were transferred to 1.5 ml tubes and centrifuged at 14,000 rpm for 10 min at 4°C to pellet debris. The pellet was discarded, and two 15 ml tubes, or 4 IPs, worth of sonicated chromatin was pooled into a fresh 15 ml tube. 3.5 ml of IP Dilution Buffer was added and mixed gently. The cross-linked DNA was pre-cleared by incubating with rotation 10 μg of rabbit IgG (Sigma-Aldrich, #I5006) for 1 hr at 4°C, followed by incubating with rotation 100 µl of Protein G agarose beads (50% v/v; Roche, #11243233001) pre-washed twice with D-PBS for 1 hr at 4°C. The samples were then centrifuged for 3 min at 3,000 rpm at 4°C and the supernatant was transferred to a fresh 15 ml tube. An aliquot of 300 µl for Input sample was transferred to a fresh 1.5 ml Eppendorf tube and stored at 4°C. The supernatant was split equally into four 15 ml tubes, each representing one sample i.e. 1 x 107 cells worth of material per tube. 10 μg of GATA6 antibody (Cell Signaling, #5851) or rabbit IgG (Sigma-Aldrich, #I5006) control was added per tube, and samples were incubated rotating overnight at 4°C. Antibody-bound chromatin was then collected using 60 µl of Protein G agarose beads (50% v/v) pre-washed twice with D-PBS for each tube by incubating with rotation for 1 hr at 4°C. Thereafter, the tubes were centrifuged for 3 min at 3,000 rpm at 4°C. The supernatant was discarded and the pellet containing the protein-DNA complexes bound onto the protein G agarose beads were kept. 500 µl of IP Wash Buffer 1 (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 50 mM NaCl, 0.1% SDS and 1% Triton X-100) was added to each tube and the tubes were vortexed. The samples were transferred to fresh 1.5 ml Eppendorf tubes and were then centrifuged at 14,000 rpm for 1 min at 4°C. The samples were washed with IP Wash Buffer 1 once more by removing the supernatant, adding 500 µl of IP Wash Buffer 1, vortexing, and centrifuging at 14,000 rpm for 1 min at 4°C. 500 µl of IP Wash Buffer 2 (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.25 M LiCl, 1% NP-40 and 1% Sodium deoxycholic acid) was then added to each tube after removing the supernatant and the tubes were vortexed and centrifuged at 14,000 rpm for 1 min at 4°C. The wash was repeated once more. Thereafter, the samples were washed twice with 500 µl of TE Buffer (10mM Tris-Cl pH 8.0, 1mM EDTA) according to the procedures above. After the supernatant was aspirated, the chromatin was eluted from the Protein G beads by washing twice with 150 µl of Elution Buffer (100 mM NaHCO3 and 1% SDS), vortexing and centrifuging at 14,000 rpm for 1 min at 4°C. The supernatants were collected and pooled in fresh 1.5 ml Eppendorf tubes. ChIP and Input DNA cross-links were reversed and RNA degraded by adding 1 μl of 1 mg/ml RNase A and 18 μl of 5M NaCl and incubating at 67°C in a heat block with shaking at 1,300 rpm overnight. Protein was degraded by adding 3 μl of 20 mg/ml Proteinase K and incubating for 3 hrs at 45°C in a heat block with shaking at 1,300 rpm. DNA was extracted using 300 µl of phenol/chloroform wash with vortexing then centrifuged at 14,000 rpm for 5 min at room temperature. The aqueous layer containing pulled down genomic DNA was transferred to fresh 1.5 ml Eppendorf tubes. 30 μl of 3M NaAc pH 5.2 (Ambion, #AM9740), 30 μg glycoblue (Ambion, #AM9516) and 750 μl of 100% ethanol were added to the samples, which were then vortexed. The samples were next incubated for at least 30 min at -80°C to precipitate the DNA. Precipitated DNA was pelleted by centrifuging at 14,000 rpm for 30 min at 4°C. The DNA pellet was then washed with ice-cold 70% ethanol and centrifuged at 14,000 rpm for 5 min at 4°C. The ethanol was removed and the pellet air dried. 70 µl of deionised water was added to Input samples whereas 30 µl of deionised water was added to ChIP samples. Library preparations were made manually using the NEBNext Ultra DNA Library Prep Kit for Illumina.
attributes.tag: ENA first public
attributes.value: 2019-07-27
attributes.tag: ENA last update
attributes.value: 2017-08-10
attributes.tag: External Id
attributes.value: SAMEA104208748
attributes.tag: INSDC center alias
attributes.value: Institute of Medical Biology
attributes.tag: INSDC center name
attributes.value: Institute of Medical Biology
attributes.tag: INSDC first public
attributes.value: 2019-07-27T17:04:09Z
attributes.tag: INSDC last update
attributes.value: 2017-08-10T10:35:29Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5959:GATA6Het_Day3_IgG.bam
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: H9
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: genotype
attributes.value: c.618_619insTGCA/+
attributes.tag: phenotype
attributes.value: definitive endoderm cell
attributes.tag: sample name
attributes.value: E-MTAB-5959:GATA6Het_Day3_IgG.bam
attributes.tag: sampling time point
attributes.value: 3