GSM8149416: Replicate I HTO; Mus musculus; RNA-Seq
Intestinal organoids were washed with PBS, resuspended in TrypLE Express Enzyme and digested for 10 minutes at 37°C. Afterwards the reaction was stopped by adding PBS + 0,5% FBS and passed through a 100µm strainer. The single cells were stained with EpCAM-BUV395 and respective Hashing antibodies (TotalSeq B). Live/dead staining was performed with Propidiumiodid. Single cells were sorted by FACS as live/dead- EpCAM+ single cells and directly processed for scRNA-seq Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 and 3' Feature Barcode Kit (Dual Index) Oligo-dT priming of full length poly adenylated mRNA, Capture Sequence 1 and 2 for Feature Barcoding library, followed by enzymatic fragmentation, P5,P7 i5,i7 are added via EndRepair, A-tailing and Adaptor Ligation