organism: Homo sapiens
description: Protocols: 50,000 cells were harvested and spin down for 5 min at 800g. Cells were grown in RPMI1640 medium supplemented with 10% foetal bovine serum, 2 mM L-Glutamine and 100 IU/ml penicillin/streptavidin at 37°C in a 5% CO2 humid atmosphere, supplemented with 10% tetracyclin-free FBS to avoid leakage. For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11. The constructed plasmid was verified by restriction digest and sequenced by Sanger DNA sequencing (GATC). Lenti-X 293T Cells (Takara, cat#632180) were transfected with the regulator vector pLVX-pEF1a-Tet3G (cat#631353 and Lenti-X Packaging Single Shots (VSV-G) (cat#631275) according to the manufacturer's instructions. The supernatant containing the lenti-virus was collected, filtered through a 0.45 µm filter and concentrated using PEG-IT. SH-EP cells were infected with the concentrated virus and 48 hours of incubation thereafter, the transduced cells were selected using 500 µg/ml G418. Individual clones were obtained by limiting dilution. After clonal expansion, the TET protein expression in each clone was checked by immunoblotting using TetR monoclonal antibody (Clone 9G9) (Clontech, cat#631131). In addition, induction of each expressing clone was tested after transduction with the pLVX-TRE3G-Luc control vector. Selected clones were transduced with lentivirus produced as described above from vector pLVX-TRE3G-Zsgreen1-IRES-hSOX11 and subsequently selected with 1 µg/ml puromycin. Each clone was treated with 1 ug/ml Doxycycline for induction of SOX11 expression or left unaffected in case of the control. ATAC-seq was performed as previously described with minor changes (Buenrostro et al. 2015). 50,000 cells were lysed and fragmented using digitonin and Tn5 transposase (Nextera DNA Library Prep Kit, cat#FC-121-1030)). Samples were purified using the DNA clean & concentrator kit from Zymo (cat#D4013). The transposased DNA fragments were amplified using the KAPA HiFi HotStart Readymix (cat#07 958 927 001) and purified using Agencourt AMPure XP beads (Beckmamn Coulter, cat#A63881). Library concentrations were measured with the Illumina Kapa Library quantification kit (Lightcycler 480 qPCR mix cat#KK4854)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA110195612
attributes.tag: INSDC center alias
attributes.value: UGent
attributes.tag: INSDC center name
attributes.value: ugent
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:58:24Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:58:24Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11898:G2_DOX_rep1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: SHEP
attributes.tag: cell_type
attributes.value: neuroblast (sensu Vertebrata)
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: neuroblastoma
attributes.tag: genotype
attributes.value: doxycycline-inducible SOX11 expression clone G2
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: bone marrow
attributes.tag: phenotype
attributes.value: SOX11 overexpression
attributes.tag: sample name
attributes.value: E-MTAB-11898:G2_DOX_rep1
attributes.tag: stimulus
attributes.value: doxycycline; 1 microgram per milliliter