organism: Homo sapiens
description: Protocols: We generated an inducible DDdCas9VP192 activator cell line in H9 human embryonic stem cells using the Sleeping beauty transposition system. The guides targeting a LEUTX promoter and enhancers were introduced by PiggyBac transposition. Three sample types were generated: Promoter only, Promoter+Enhancer1, Promoter+Enhancer2. Cells were collected at 0 h, 24 h, 48 h, and 72 h after doxycycline treatment. Uninduced cells were used as controls. The RNA was extracted using NucleoSpin RNA Plus kit (Macherey-Nagel) according manufacturer's instructions. We used 20 ng of RNA to generate a 48-plex RNA-seq library using a modified STRT method with unique molecular identifiers (UMIs) (Islam et al., 2011, 2014). Briefly, RNA samples were placed in a 48-well plate, and a universal primer, template-switching oligo-nucleotides, and a well-specific 6 bp barcode sequence (for sample identification) were added to each well (Krjutškov et al., 2016). We pooled the synthesized cDNAs into one library, performed fragmentation to 200–400bp using an M220 Focused-ultrasonicator (Covaris), captured the 5'-prime fragments, added an adapter, and amplified the targets by PCR.
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: SAMEA9089016
attributes.tag: INSDC center alias
attributes.value: Department of Biosciences and Nutrition, Karolinska Institutet
attributes.tag: INSDC center name
attributes.value: Department of Biosciences and Nutrition, Karolinska Institutet
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:56:50Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:56:50Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10539:LEUTX_47
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: developmental stage
attributes.value: embryo stage
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: DDdCas9VP192
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: blastocyst
attributes.tag: phenotype
attributes.value: doxycycline-induced activation of LEUTX promoter and enhancer2
attributes.tag: replicate
attributes.value: 3
attributes.tag: sample name
attributes.value: E-MTAB-10539:LEUTX_47
attributes.tag: sex
attributes.value: female