NextSeq 500 sequencing; Whole culture RNA-sequencing of transient DUX4-expressed human embryonic stem cells
Whole culture RNA-sequencing of transient DUX4-expressed human embryonic stem cells
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.