characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-12-31T00:58:40Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-12-31T00:58:40Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111529184
characteristics.characteristic: External Id
characteristics.text: Philipps University Marburg, Bioinformatics Core Facility, Center for Tumor- and Immune Biology
characteristics.characteristic: INSDC center name
characteristics.text: 2022-12-31T00:58:40Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-12-31T00:58:40Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13639165
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12344:TA235
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: IMR-32
characteristics.characteristic: cell_line
characteristics.text: neuroblast (sensu Vertebrata)
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Cells were harvested with a spatula. 2 Lysis, sonication, preclearing, addition of 10% 3T3 chromatin (untreated, same for all samples), followed by the nucleic acid extraction protocol Cells were grown in DMEM (high Glucose), 10%FBS, 1%PenStrep at standard conditions. ISX or DMSO control was added, cells were harvested 8h later for ChIP. was performed with 1% formaldehyde in PBS for 10 min at room temperature followed by quenching with 125 mM glycin for 5 min. Cells were washed with ice-cold PBS twice and harvested using a cell scraper. The pellet was lysed in hypotonic buffer L1 (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% (v/v) NP40) with protease inhibitor mix (Sigma, no. P8340, 1:1,000) for 20-40 min on ice. Nuclei were resuspended in ChIP RIPA buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40 (v/v), 1% sodium deoxycholate (w/v), 1 mM EDTA) supplemented with 1:1,000 protease inhibitor mix (Sigma), incubated on ice for 10-20 min and sheared with a Branson S250D Sonifier (Branson Ultrasonics) using a microtip in 1 ml aliquots in 15 ml conical tubes. 52 pulses of 1 s, 4 s pause, 20% amplitude were applied with cooling of the sample in an ice-ethanol mixture or in a 15 ml tube cooler (Active Motif, no. 53077). A 15 min 20,000×g supernatant was precleared with 10 µg of IgG coupled to 100 µl of blocked sepharose slurry (see below) for 45 min at 4 °C with agitation. IP was carried out with 300 µl of precleared chromatin, equivalent to 3-8 × 106 cells. For MIRA, an aliquot of the sample was reverted as described below, purified on a Qiagen PCR purification column (no. 28106), and 250 ng of DNA were used according to the manufacturer’s instructions (Methylcollector Ultra, Active Motif, no. 55005). ChIP was performed and evaluated as described using 4 µg per sample of the respective antibodies. For precipitation, a mixture of protein A and protein G sepharose (GE Healthcare life sciences, no. 1752800 and no. 1706180) was washed twice with ChIP RIPA buffer and blocked with 1 g/l BSA and 0.4 g/l sonicated salmon sperm DNA (Life Technologies no. 15632011) overnight. 50 µl of blocked bead slurry (1:1 volume ratio with liquid phase) were used per IP. Samples were washed once in buffer I (20 mM Tris pH 8.1; 150 mM NaCl; 1% (v/v) Triton X-100; 0.1% (w/v) SDS; 2 mM EDTA), once in buffer II (20 mM Tris pH 8.1; 500 mM NaCl; 1% (v/v) Triton X-100; 0.1% (w/v) SDS; 2 mM EDTA), twice in buffer III (10 mM Tris pH 8.1; 250 mM LiCl; 1% (v/v) NP40; 1% (w/v) sodium deoxycholate; 1 mM EDTA) on ice, and twice in Qiagen buffer EB (no. 19086) at room temperature. Immune complexes were eluted twice with 100 mM NaHCO3 and 1% SDS (w/v) under agitation. Eluates were incubated overnight at 65 °C after adding 10 µg of RNase A and 20 µg of proteinase K in the presence of 180 mM NaCl, 35 mM Tris-HCl pH 6.8, and 9 mM EDTA. An input sample representing 1% of the chromatin used per IP was reverted in parallel. Samples were purified using the Qiagen PCR purification kit according to the manufacturer’s instructions, except that DNA-binding lipids were removed by washing the matrix twice with pure methanol. previous to the final washing step with the buffer included in the kit. Libraries were synthesized from 1-2 ng of genomic DNA using the MicroPlex kit (Diagenode, no. C05010011) according to the manufacturer’s instructions.
characteristics.characteristic: description
characteristics.text: infant
characteristics.characteristic: developmental stage
characteristics.text: neuroblastoma
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: brain
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: TA235
characteristics.characteristic: title