GSM7732260: KO, replicate 1, scRNAseq; Mus musculus; RNA-Seq
A 2 cm-long fragment of the proximal intestine was extracted from mice, washed with PBS and cut longitudinally. This fragment was incubated for 20 minutes at 4°C in a DPBS buffer supplemented with 30 mM EDTA and 1,5 mM DTT, then incubated for 10 minutes at 37°C in a DPBS-EDTA buffer (30mM). Cells were harvested, centrifuged at 300 g for 5 minutes at 4°C and washed with DPBS-FBS 10%. After centrifugation, samples were dissociated into single cell suspensions by incubation in HBSS-dispase (0,8 mg/mL) for 12 minutes at 37°C. Single cell solutions were sequentially passed through 70- and 40-mm strainers. Red blood cells were lysed (#130-094-183, Miltenyi Biotec) for 2 minutes at room temperature. Cells were counted and 10000 cells were sent for single cell RNA sequencing experiments. For library preparation and sequencing, 10000 cells were injected for each sample in 4 different reactions into the Chromium Single Cell Controller following the protocol of the Chromium NextGEM Single Cell 3' V3.1 Kit (10x Genomics). The protocol was followed according to the manufacturer's guidelines.The quality control of DNA library was performed on Qiaxcel (QIAgen) and quantification by qPCR (KAPA SYBR Fast & LQ Stds+Primer (Illumina). All Single cell 3' Gene expression libraries were sequenced on a Novaseq 6000 System (Illumina) on a S1 flowcell.