GSM1876367: MabATCC_ribo_R2; Mycobacteroides abscessus ATCC 19977; RNA-Seq
The bacterial pellets were re-suspended in lysis buffer (100 mg/mL chloramphenicol, 70 mM KCl, 10 mM MgCl2, 10 mM Tris-HCl [pH 7.4]) and physically disrupted by two passages through a French Press (10,000 psi), followed by centrifugation to remove intact cells and cellular debris. The clarified lysates were digested with MNase and RNaseI. Ribosome protected fragments were recovered by sedimentation through a 1 M sucrose cushion, size selected by gel purification and prepared for high throughput sequencing (doi: 10.1038/nprot.2012.086). Briefly, 3' termini of ribosome protected fragments were dephosphorylated with polynucleotide kinase (PNK, New England Biolabs) allowing for ligation of a 3' RNA TruSeq small RNA adapter (Illumina Inc.). The 5' termini were then phosphorylated by PNK and 5' RNA TruSeq small RNA adapter was ligated. Following reverse transcription, the ribosome protected fragments were amplified by PCR and PCR products of expected size were purified from a Tris-Borate-EDTA gel.
GEO Accession
GSM1876367