GSM8678731: Gpx4 KO biol rep 1; Mus musculus; RNA-Seq
After stimulation at 37°C for 8h, cell culture medium was discarded and cells were rinsed once with PBS. RNA isolation was performed according to manufacturer's instructions using a RNeasy mini kit (Qiagen, 74104). For single cell isolation, the small intestine (SI) was cleaned of adhering stool/tissue. Afterwards the middle 60 % of the SI was flushed with ice cold PBS. The SI was then opened longitudinally and washed again with ice cold PBS. Thereafter, the tissue was cut into small pieces and digested in 50ml PBS containing 2mM EDTA. Samples were shaken regularly for the next 30min. The supernatant was collected and remaining tissue pieces were dissolved in ice cold PBS and shaken again. This step was repeated 4 times. Supernatant was then centrifuged for 5 min at 300 g at 4°C and the resulting cell pellet was dissolved in trypsin + PBS (1:1), vortexed gently and dissolved in DMEM containing FCS. Accordingly, cells were counted and incubated with beads for live dead separation using a dead cell removal kit (Miltenyi Biotec, 130-090-101) according to the manufacturer's recommendations. The viable cells were collected and centrifuged at 300 g and 4°C for 5 min. Pellets were dissolved in 100 µl ice-cold PBS containing 0,4 % BSA. Bulk RNA-seq: Purified total RNA was processed using the QuantSeq 3' mRNA-Seq Library Prep method (Lexogen, Vienna Biocenter). Single cell suspensions were processed with a Chromium single cell controller (10xGenomics) and Chromium Next GEM Single Cell 3' Kit v3.1 chemistry, targeting 8000 cells per sample.