GSM8579954: NPM6_DOX; Homo sapiens; RNA-Seq
The tissue samples were incubated in 20mM EDTA-PBS while rocking in a cold room for 90 minutes. Every 30 minutes, the tubes containing the intestinal tissues were shaken vigorously for one minute to collect dissociated epithelial fractions. After 90 minutes, the three collections from each tissue were combined and dissociated into single cells using TrypLE for one minute at 37°C.The single-cell suspensions in FACS buffer (containing 1% glucose, 10mM HEPES, 10μM Y-27632, 1mM N-acetyl-L-cysteine, and 2% FBS in DPBS) were passed through a 40μm filter. The cells were then stained with anti-mouse CD326 (EpCam, Key Resources Table). Live EpCam+ epithelial cells were sorted using a Symphony S6 II cell sorter in FACS buffer. For each sample, 10,000 cells were loaded into each channel of the Chromium Controller cartridge to produce droplets. Beads-in-emulsion (GEMs) were transferred, and GEM reverse transcription (GEM-RT) was performed in droplets via PCR incubation. After the post-GEM-RT reaction, first-strand cDNA was purified. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. TruSeq Read 1 (read 1 primer sequence) was added during GEM incubation. A sample index and TruSeq Read 2 (read 2 primer sequence) were added through end-repair, A-tailing, adaptor ligation, and PCR. The final libraries were assessed using an Agilent Technology 2100 Bioanalyzer and sequenced on an Illumina NovaSeq sequencer.