RNeasy Plus Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Pre-treatment with lysostaphin	To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	Include DNAse treatment for 15-20min. Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. Use water to elute the RNA that is warmed to ~60`C

Upstream tips
Pre-treatment with lysostaphin
Protocol tips
To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
Include DNAse treatment for 15-20min. Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. Use water to elute the RNA that is warmed to ~60`C

Publication protocol

Samples of biofilm cells were collected as described above into 3 mL of RNAprotect Bacteria Reagent (Qiagen). Following 10-minute room temperature incubation, mRNA isolation (RNeasy Mini Kit, Qiagen) was carried out using approximately 3 × 108 cells according to the manufacturer’s protocol, with the following modifications. For biofilm experiments, P. aeruginosa cells were treated with 400 μg/mL lysozyme in TE for 5 min at room temperature prior to RNeasy RNA isolation. For planktonic and co-culture experiments, cells were treated with 400 μg/mL lysozyme and 100 μg/mL lysostaphin in TE for 30 min at 37 °C prior to RNeasy RNA isolation. The RNeasy elution step was also modified to use 75 uL of 1X TE (10 mM Tris-HCl 1 mM EDTA) buffer to ensure downstream stability of the RNA during the RNA-seq library preparation. The RNA was subsequently subjected to DNase treatment (TURBO DNA-free DNase Treatment, Ambion) according to the manufacturer’s protocol. RNA quantity and quality were assessed as described below.

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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Plus Mini Kit below.

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