GSM7439492
Subsequently 10 million cells were methanol fixated according to 10X Genomics Methanol Fixation of Cells for Single Cell RNA Sequencing protocol User guide CG000136 Rev E, using a mix of two RNAse inhibitors (RNase Inhibitor, Thermo Fisher, Carlsbad, CA, USA and RNasin® Plus RNase Inhibitor, Promega, Madison, WI, USA). Samples were stored < 6 weeks before proceeding with Chromium run. Cells were rehydrated following the MetOH fixation protocol and processed for loading following the Chromium Single Cell 3´Reagent Kits v3 User guide CG000183 Rev C. Mouse cells were prepared into pools together with human cells (not part of this study) and mouse cell singlets separated in data analysis step. Using 10X GemCode Technology, the poly-A transcripts are barcoded with an Illumina R1 sequence, a 16 bp 10X barcode and a 10 bp Unique Molecular Identifier (UMI). 13 cycles of PCR were used to amplify the cDNA. Sequencing was performed at Novogene UK sequencing core facility aiming at 50 000 reads per cell depth.