description: Protocols: Doxycycline-induced and control DUX4-TetOn hESCs were collected immediately, 24 h, and 48 h after 15 min, 30 min of doxycycline, or without doxycycline treatment. Naïve H9 hESCs were collected by hand picking the colonies from the cell culture dishes, using sterile needles. Conventional primed H9 hESCs were collected by washing the cells once with PBS and lysing the culture according to the NucleoSpin RNA kit protocol. For the NaPi2b+/- cells, the DUX4-TetOn hESCs were sorted by FACS at 6 h after induction and collected after a further 6 h of culture. Total RNA was isolated using NucleoSpin RNA kit (Macherey Nagel) according to the manufacturer's protocol. 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: 2022-06-30
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-06-30
attributes.tag: External Id
attributes.value: SAMEA13166893
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: 2022-06-30T00:18:53Z
attributes.tag: INSDC last update
attributes.value: 2022-06-30T00:18:53Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10569_2:dox unsorted_3
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: fraction
attributes.value: unsorted
attributes.tag: genotype
attributes.value: TetOn DUX4-full-length
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: sample name
attributes.value: E-MTAB-10569_2:dox unsorted_3
attributes.tag: stimulus
attributes.value: 15 min doxycycline-induction