Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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ATAC-seq to detect open chromatin sites in a selection of ovarian cancer cell lines

ABSTRACT: ATAC-seq was performed in the ovarian cancer cell lines COV-434, ES-2, Kuramochi, OAW28, OAW42, OVISE, SKOV-3, TOV-112D. The aim of this experiment was to detect open chromatin sites. Enhancers are associated with open chromatin sites flanked by histones with H3K4me1 and H3K27ac marks. Therefore, the combination of this ATAC-seq experiment with histone ChIP-seq is complementary for the detection of enhancers. Omni-ATAC-seq was performed as described by Corces et al. 2017 with some modifications, which are described in detail in the protocol.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

SUBMITTER: Leonie Smeenk

PROVIDER: E-MTAB-14770 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Patients received standardized coartem therapy. Briefly, each pill contains 20 mg of artemether and 120 mg of lumefantrine. The dose and total coarse of tablets is based on the patients body weight. Blood was drawn from patients prior to anti-malarial treatment (day 0) and after treatment (day42 for children and day 28 for adults). Peripheral Blood Mononuclear Cells were isolated from all patients. Briefly, for PBMC isolation, whole blood from was diluted 1:2 in sterile PBS and overlaid on Ficoll Paque-PLUS in 50 mL conical tubes and centrifuged 800xg for 15 minutes at 25°C with no brake. Resulting buffy coats were collected and washed twice in RPMI 1640 medium, and then cells were treated with Red Blood Cell Lysis Buffer for 3 minutes at room temperature. Cells were washed in RPMI and counted on a hemacytometer. Monocytes were purified from PBMC using a miltenyi Pan Monocyte Isolation negative selection kit according to the manufacturer's instructions. For each sample. 50,000 viable cells were pelleted and lysed with cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and incubated in ice for 3 minutes. The lysaste was washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin. Nuclei were pelleted at 500 RCF for 10 min at 4 degrees celcius. The supernatant was discarded and the pellet was resuspended in 50 uL of transposition mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS,0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O). The reaction was incubated for 30 minutes in a thermomixer with 1000 RPM mixing. Zymo DNA Clean and concentrator kit (cat# D4014) was used as a cleanup step. The DNA was amplified at 98 degrees C for 30 seconds, then 13 cycles of: 98 degrees C for 10 seconds, 63 degrees C for 30 seconds, 72 degrees C for 1 minute.The library was evaluated in an Agilent TapeStation system and the DNA concentration evaluated by Qubit. Sequencing was performed on Illumina Nextseq 500 with Buffer cartridge v2: Ref: 15057941, High output reagent cartridge v2: Ref: 15057934, NextSeq accessory Box v2: Ref: 15058251, and High output Flow Cell cartridge v2.5: Ref: 20022408

Project description:Erythrocytes were infected with Plasmodium chabaudi chabaudi AS strain expressing GFP. After 2 or 3 passages, a suspension of 100,000 infected erythrocytes in 1X-PBS (adjusted volume of 200µl) were injected i.p. in wild-type C57BL/6j mice and Homozygous mutant Ighm tm1Cgn mice (lacking mature B-cells). As a control, wild-type C57BL/6j mice and Homozygous mutant Ighm tm1Cgn mice were injected i.p with PBS. There were 3 mice (biological replicates) for each of the 4 experimental conditions. Parasitemia was monitored by flow cytometry and PCR weekly. 117 days post infection, mice were euthanized in CO2 chamber and femur and tibia bones were collected in 1X-HBSS/2% FBS. The cells were removed from the bones and the erythrocytes lysed by hypotonic lysis. Cells (20 million) were antibody stained and the 100K cells (Live/CD11b+/Ly6G-/F4/80+) were sorted in 1X-HBSS/20% FBS. For each sample. 50,000 viable cells were pelleted and lysed with cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and incubated in ice for 3 minutes. The lysaste was washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin. Nuclei were pelleted at 500 RCF for 10 min at 4 degrees celcius. The supernatant was discarded and the pellet was resuspended in 50 uL of transposition mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O). The reaction was incubated for 30 minutes in a thermomixer with 1000 RPM mixing. Zymo DNA Clean and concentrator kit (cat# D4014) was used as a cleanup step. The DNA was amplified at 98 degrees C for 30 seconds, then 13 cycles of: 98 degrees C for 10 seconds, 63 degrees C for 30 seconds, 72 degrees C for 1 minute.The library was evaluated in an Agilent TapeStation system and the DNA concentration evaluated by Qubit. Sequencing was performed on Illumina Nextseq 500 with Buffer cartridge v2: Ref: 15057941, High output reagent cartridge v2: Ref: 15057934, NextSeq accessory Box v2: Ref: 15058251, and High output Flow Cell cartridge v2.5: Ref: 20022408

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ATAC-seq to detect open chromatin sites in a selection of ovarian cancer cell lines

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