GSM9116790: null strain replicate; Bacillus subtilis subsp. subtilis str. 168; RNA-Seq
At the indicated timepoints wildtype and ppGpp0 cells were fixed in 1% formaldehyde at room temperature for 30 minutes. After fixation cells were pelleted by centrifugation at 6000 RPM for 3 min at room temperature and washed with 0.02% saline sodium citrate (SSC, Invitrogen). The cell were pelleted again and resuspended in 300 µl MAAM mix (4:1 V:V dilution of methanol to glacial acetic acid) and stored at 20 oC until processing. ProBac-seq sample processing was done as described previously (see McNulty et al Nat Micro 2023). Briefly, samples were pelleted and resuspended in PBS to remove MAAM. Following PBS wash to remove MAAM the cells were incubated with lysozyme (Readylyse, Epicenter biotechnology) for 30 minutes in room temperature for cell wall hydrolysis. After an additional pelleting step and PBS wash the samples were further permeabilized by a 5 minute incubation in PBS-Tween (PBS with 1% final concentration of Tween 20 detergent) and a subsequent wash in PBS to remove the detergent. Permeabilized cells were resuspended in 100 ul of probe binding buffer (5 × SSC, 30% formamide, 9 mM citric acid (pH 6.0), 0.1% Tween 20, 50 ug ml−1 heparin and 10% low molecular weight dextran sulfate purchased from Molecular Instruments inc. as Probe Binding Buffer – Cells in Suspension). A transcriptome-wide probe-set containing approximately 30,000 mRNA binding probes designed against the B. subtilis genome (see McNulty et al Nat Micro 2023) was added to each sample before placing the samples to incubate overnight at 50oC.  After overnight incubation unbound probes were removed by a series of 7 washes with probe wash buffer (5 × SSC, 30% formamide, 9 mM citric acid pH 6.0, 0.1% Tween 20 and 50 ug ml-1 heparin Molecular Instruments inc.) and 2 final washes in PBS. Probed and washed cells were enumerated using a flow-cytometer and approximately 10,000 cells per condition were processed using the ProBac-Seq protocols as described previously (see McNulty et al Nat Micro 2023). In short, 51 ul cell suspensions in PBS were mixed with 19 ul ddPCR master mix (Biorad), 3 ul in-droplet PCR primer, 2 ul reducing agent (10X genomics) to provide a 75ul reaction mix. 70 ul of this reaction mixture was loaded onto 10X Genomics microfluidic chip-G devices alongside 10X barcoding beads and emulsion oil. The devices were run using a 10X chromium controller and emulsions were processed to produce barcoded libraries as described previously.