Comparative Sample Disintegration Techniques

We compared different sample disintegration techniques, namely the laboratory grinding mill Micro-Dismembrator (Sartorius, Göttingen, Germany), the TissueLyser (Qiagen, Hilden, Germany), and the cryoPREP impactor (Covaris, Brighton, UK). Sample disintegration using the Micro-Dismembrator was essentially performed as described^{27 (link)}. Using the TissueLyser, tubes prepared with the sample material, a steel grinding ball and 200–1000 µl AL buffer (Qiagen, Hilden, Germany) were shaken for 150 s with a frequency of 30 Hz as previously described^{50 (link),51 (link)}. With a Micro-Dismembrator (Sartorius, Göttingen, Germany), the samples were ground frozen in liquid nitrogen for 2 min at 2000 rpm in a 3 ml PTFE shaking flask with a 10 mm stainless steel ball and the frozen homogenate was further processed according to the detailed protocol (Supplementary File 1, from step 9). The cryoPREP protocol is given in detail in Supplementary File 1 (Procedure, steps 1–10). RNA was extracted following the detailed protocol and quality was checked with a Bioanalyzer (Agilent) using a RNA 6000 pico assay according to the manufacturer’s instructions. DNA from Mycobacteria containing samples was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and quantified regarding the content of mycobacterial DNA via real-time PCR (insertions element IS900^{52 (link)}).