GSM9410116: 20minutes_with_opp; Bacillus subtilis; RNA-Seq
Single cell Library was performed according to specifications of McNulty et al bacterial scRNAseq protocols (single cell 3' v2 protocol, 10x Genomics). Briefly, Single cell partitioning, barcoding, and cDNA library generation was achieved using the 10X Genomics Chromium Controller with the Chromium Single Cell 3' Reagents Kit (v2 chemistry) as described by 10X Genomics (https://support.10Xgenomics.com/single cell-gene-expression/index/doc/user-guide-chromium-single cell-3-reagent-kits-user-guide-v2-chemistry). The protocol was modified to achieve bacterial scRNA-seq. For GEM generation (10X microfluidic encapsulation), a master mix containing the following reagents (per rxn, not accounting for excess volume) was prepared: 33 µL of 4X ddPCR Multiplex Supermix (BioRad), 4 µL of custom primer (10 µM), 2.4 µL additive A (10X Genomics), 26.8 µL dH2O. All other reagents specified by 10X Genomics were omitted. Prepared cell samples were washed 3X in PBS and diluted to 1000 cells/µL before loading on the microfluidic chip (“Chip A Single Cell”) with a targeted cell recovery of 10,000 cells.