scRNA-seq: Bag 7 Day 17
For analysis by 10X Genomics, tubes were defrosted and gently mixed, and 1.7 ml of the samples were transferred into an Eppendorf Lowbind tube and centrifuged at 4C for 3 min at 3000g. The PBS/methanol mix was discarded and replaced by 400 l of PBS. Cell concentration was measured using an iCyt Eclipse flow cytometer (SONY), based on forward scatter. Cell concentration ranged from 1044 cells ml-1 to 9855 cells ml-1. All concentrations were brought to 1000 cells ml-1 to target 7000 cells recovery, according to the 10X Genomics Cell Suspension Volume Calculator Table provided in the user guide. Cellular suspension was loaded onto Next GEM Chip G targeting 7000 cells and then ran on a Chromium Controller instrument to generate GEM emulsion (10x Genomics). Single-cell 3 RNA-seq libraries were generated according to the manufacturers protocol (10x Genomics Chromium Single Cell 3 Reagent Kit User Guide v3/v3.1 Chemistry) on different occasions: B4T19 and B7T17 in January 2020 and B3T15, B3T20, B4T13, B4T15, B4T20, B6T17, B7T16, and B7T18 in August 2020 with 12 cycles for cDNA amplification and 15 cycles for library amplification. Library concentrations and quality were measured using the Qubit dsDNA High Sensitivity Assay kit (Life Technologies, Carlsbad, CA). Libraries were pooled according to targeted cell number, aiming for a minimum 20,000 reads per cell. Pooled libraries were sequenced on using NextSeq 500 High Output kit (75 cycles).