GSM3357922: JS96_TCACAC_trimmed; Mus musculus; RNA-Seq
On day 2.5 after culture, each 200 μl cultured well was mixed well, then 100 µL of the contents transferred manually to a separate new well, and given approximately 30-60 minutes to settle. The 96-well plates containing sister wells derived from each single UBC-GFP LSK clone were imaged on a Nikon TiE live cell imaging system. Cells were maintained at 37 °C in a humidified atmosphere and 5% CO2. Images were acquired in DIC and fluorescence using a 4x 0.13NA plan neofluor objective, and GFP filter (FITC, ex 465-495, DM 505, em 515-555). A Z-stack of 150 μm, with interval of 50um was performed and, the best focus plane was automatically selected for analysis using an offline Metamorph software (v 7.8.2.0) licence and method for automatic segmentation and cell count. Cell count script parameters, region size and diameter, were changed per experiment, on the basis of visual inspection of the 100 single cell controls. After imaging, cell cultures were incubated in 37 °C and at 5% CO2 for 4.5 days. Depending on cell abundance 1 – 10 cells per clone were FACS sorted for RNA sequencing. Transcriptome libraries were generated according to the CEL-Seq protocol with the exception that all column purifications were replaced by magnetic bead purifications using AMPure XP beads (Beckman Coulter A63880). Libraries were sequenced on the Illumina NextSeq500 sequencer using a 75-cycle kit.
GEO
GSM3357922