GSM9543212: Pi; Prevotella intermedia; RNA-Seq
500,000 bacterial cells were pelleted by centrifugation at 4 °C. The pellet was resuspended in 2 mL of ice-cold 4% formaldehyde (Sigma, #47608), followed by an overnight fixation with shaking at 4 °C. After centrifugation at 4000 rpm for 5 min at 4 °C, the fixative was removed. The pellet was washed twice with 1 mL of PBS-TRI (1× PBS containing 0.1% Tween-20 and 0.1 U/mL Murine RNase Inhibitor (Vazyme, #R301), and then resuspended in 200 μL of PBS-TRI (1× PBS with 0.1% Tween-20 and 0.2 U/mL Murine RNase Inhibitor (Vazyme, #R301). The cell concentration was determined using a MoxiTM Z cell counter, and the suspension was diluted according to the manufacturer's protocol to achieve a single-cell suspension. Libraries were prepared from bacterial cells using the VITAPilote® kit (M20 Genomics, #R20114124) according to the manufacturer's protocol.