GSM9567173: 15653_Mtb_LDA_Day_11; Mycobacterium tuberculosis str. Erdman = ATCC 35801; RNA-Seq
Murine tissue samples were snap frozen in 7 mL Precellys tubes (Bertin) under liquid nitrogen. To each tissue sample, 1.5 mL GTC-TCEP buffer was added and samples were homogenized at 7,200 rpm for 16s using a Precellys Evolution (Bertin). Homogenates were incubated at room temperature for 5 minutes while protected from light. Unlysed mycobacterial cells were pelleted at 10,000g for 3 minutes, then resuspended in 0.8 mL RLT+ BME buffer (Qiagen, TCEP concentration 0.75 mM). Mycobacterial cells were lysed on a Precellys Evolution (Bertin) at 6,500 rpm for 30s three times, resting on ice for 5 minutes each time, and stored at -80C. After storage at -80C, murine lysates were thawed, and RNA was purified using the Maxwell simplyRNA tissue kit (murine samples) using the Maxwell RSC instrument following the manufacturer's instructions with the following modifications. Additional kit DNase was added at twice the manufacturer's recommended amount. Purified RNA was quantified using the Quantifluor RNA system (Promega). Murine RNA was purified with Maxwell simplyRNA tissue kit while bacterial RNA was prepared for SEARCH-TB using Illumina's AmpliSeq for Illumina Custom and Community RNA Panels kit.