title: single call RNAseq for E.coli BL21 wildtype
design: Bacterial single-cell RNA sequencing was carried out on a commercially available platform (M20 Genomics Company) (Xu et al., 2023). In brief, half a million bacterium cells were collected by centrifuging at 4. The supernatant was removed and cell pellets were resuspended in 2 ml ice-cold 4% formaldehyde (Sigma, 47608) and incubated with shaking overnight at 4. The fixative was removed by centrifuging at 4000 rpm for 5 min at 4 and cells were washed twice with 1 mL PBS-TRI (1x PBS supplemented with 0.1% Tween-20 and 0.1 U/mL Murine RNase inhibitor (Vazyme, R301)). The supernatant was removed and cells were resuspended in 200 l PBS-TRI (1x PBS supplemented with 0.1% Tween-20 and 0.2 U/mL Murine RNase inhibitor (Vazyme, R 301)). The cell suspensions were counted with a Moxi cell counter and diluted according to the manufacturers instructions to obtain single-cell. The bacterial single-cell RNA-Seq library was prepared according to the protocol of VITAPilote kit(M20 Genomics, R20114124). In situ reverse transcription of bacteria was performed with random primers and the resulting cDNA fragment was added with adaptor. The droplet barcoding for a single bacterium was performed on VITACruiser Single Cell Partitioning System(M20 Genomics, Hangzhou, China). Bacteria, DNA extension reaction mix, and hydrogel barcoded beads were encapsulated using the VITACruiser. The aqueous phase containing cDNAs was purified with magnetic beads. The cDNAs were amplified by PCR, and purified with magnetic beads. All products were pooled to construct a standard sequencing library. Sequencing was done on a PE150 (Illumina)
library_name: B1211DE31