GSM6914669: Mab Control Rep 3; Mycobacteroides abscessus ATCC 19977; RNA-Seq
RNA was harvested through the addition of 2 volumes of RNA protect (Qiagen), then centrifuged for 4 minutes at 10,000xg at 4C. Supernatant was poured off and pellets stored at -80°C. Next day samples were thawed out and re-suspended in 500µL RLT + 1% betamercaptoethanol and transferred to Lysing Matrix B (VWR, Pittsburgh PA). Samples were placed into a Bead Mill 4 homogenizer (Fisher Scientific, Atlanta GA) and homogenized 4 times for 40 sec at 4.5m/s with 1 min of wet ice cooling between each round of homogenization. Lysate was spun down at 8000g for 2 minutes and supernatant removed to clean centrifuge tube. Subsequent extraction of RNA was performed according to the RNAeasy extraction kit (Qiagen). RNA was eluted in 40µL of elution buffer yielding a typical concentration of 400-500ng/µL. RNA samples were then DNase treated to remove residual genomic DNA from samples. This was performed using the Genomic DNA Clean and Concentrator Kit (Zymo Research, Irvine CA). Samples were eluted in 30-45µL of RNase free water, yields ranging from 100-250ng/µL. Libraries were prepared from total RNA with the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer's instructions (Illumina, PN 20020595). Libraries were analyzed for insert size distribution using the 2100 BioAnalyzer High Sensitivity kit (Agilent). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (ThermoFisher) and by low pass sequencing with a MiSeq nano kit (Illumina). Paired end 100 cycle sequencing was performed on a NovaSeq 6000 (Illumina) or HiSeq 2500 by the St. Jude Children's Research Hospital sequencing core at a depth of 100M reads/sample.