GSM5261802: CreTW; Mus musculus; RNA-Seq
Isolated PFE cartilage was chopped and incubated at 37°C under constant rotation (HBMSUV-14 Hybaid Hyberization Oven Maxi 14, Mount Holly, USA) using collagenase P solution (1.61U/ml collagenase P (Roche Diagnostics GmbH; Mannheim; Germany) in DMEM F12 (Life Techologies Europe B.V., Bleiswijk, Netherlands) containing 1% P/S (Life Techologies Europe B.V., Bleiswijk; Netherlands), 10% FCS (Biochrome GmbH, Berlin, Germany) and 0.1755mg/ml cystein (Sigma-Aldrich, St. Louis, USA). After 18 h, the cell-suspension was transferred through a 40µm cell strainer and washed with 1.5% BSA (Serva Electrophoresis GmbH, Heidelberg, Germany) in PBS (Life Technologies Limited, Paisley, UK). Cells were centrifuged at 400g for 5 min (Eppendorf Centrifuge 5415 R, Hamburg, Germany), resuspended in 200µl 1.5% BSA/PBS (200cells/µl) and cell viability (>90%) was confirmed by typan blue staining cDNA was prepared according to the 10x Genomics Chromium Single Cell 3' Reagent Kit User Guide (v3  Chemistry) from the pool of  ~1.000 cells . At this point, all cDNA fragments carried the 16nt cellular barcode and the 12nt long Unique Molecular Identifiers (UMI) at the Poly (dT) end. 25% (10 µL) of this cDNA solution continued with the original 10x Genomics protocol to create an Illumina 3´mRNA library with P5 and P7 Illumina adapters.  Sequencing was done on Illumina NovaSeq6000 systems with a two step approach (to validate cell numbers, UMI count and saturation) Single cell RNA Sequencing
GEO
GSM5261802