Chromium single cell 3 reagents kit

The 10x Genomics Chromium Single Cell 3′ Reagents Kit user guide (https://support.10xgenomics.com) was used to prepare the single-cell suspension. The appropriate volume of each sample was diluted to recover 10,000 cells. Subsequently, the single-cell suspension, gel beads, and oils were added to the 10X Genomics single-cell chip. After droplet generation, samples were transferred into PCR tubes and we performed reverse transcription using a ProFlex PCR Thermocycler System (Applied Biosystems, Foster City, CA). After reverse transcription, cDNA was recovered using a recovery agent, provided by 10X Genomics, followed by silane DynaBead clean-up as outlined in the user guide.

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/permalink/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) and 26.8 µl dH₂O. All other reagents specified by 10X Genomics were omitted. Prepared cell samples were washed three times in PBS and diluted to 1,000 cells µl⁻¹ before loading on the microfluidic chip (‘Chip A Single Cell’) with a targeted cell recovery of 10,000 cells. Our recovery was lower than the expected cell number and we attribute this to the difficulty in obtaining accurate cell number measurements when working with small numbers of fixed bacterial cells.