title: Illumina NovaSeq 6000 paired end sequencing; Single-cell RNA-sequencing of transient DUX4-expressed human embryonic stem cells
design: Single-cell RNA-sequencing of transient DUX4-expressed human embryonic stem cells
library_construction_protocol: Doxycycline-pulsed and control DUX4-TetOn hESCs were collected for scRNA-seq after 6 h, 12 h, 24 h, and 48 h incubation times after a 15 min doxycycline or without doxycycline treatment. The cells were washed once with PBS and incubated with TrypLE Express (Thermo Fisher Scientific) for 4 min. TrypLE was diluted with Essential 8 medium and the cell suspensions were filtered through 40 µm Cell Strainers. Cell suspensions were centrifuged at 400 rcf for 8 min. Cell pellets were resuspended each in 100 µl of Dead Cell Removal Kit microbeads (Miltenyi Biotec) and incubated at room temperature for 15 min. After incubation, each cell-microbead suspension was gently resuspended to 800 µl of freshly prepared 1× Binding buffer. Cell suspensions were pipetted to magnetic MS columns (Miltenyi Biotec) 500 µl at a time and let flow through. The columns were washed three times with 1× Binding buffer. The cell suspensions were centrifuged at 400 rcf for 5 min, and the pellets were resuspended each in 400 µl of 10x Genomics sample buffer. The cells were counted and the volumes were adjusted to approximately 1,200 cells/µl of suspension.  RNA extraction was performed per in chip inside 10x chromium controller. Libraries were prepared using Chromium Next GEM Single Cell 3' Gene Expression version 3.1 chemistry.