GSM3673863: Smeg_hypoxia 24 h_polyphosphatase Rep1; Mycolicibacterium smegmatis; RNA-Seq
RNAseq 5' end mapping library. 5' triphosphates were converted to 5' monophosphates by 5' Polyphosphatase before adapters were ligated to RNA 5' ends. RNA was extracted as follows: frozen cultures stored at −80◦C were thawed on ice and centrifuged at 4,000 rpm for 5 min at 4◦C. The pellets were resuspended in 1 ml Trizol  Life Technologies) and placed in tubes containing Lysing Matrix B (MP Bio). Cells were lysed by bead-beating twice for 40 s at 9 m/sec in a FastPrep 5G instrument (MP Bio). 300 µl chloroform was added and samples were centrifuged for 15 min at 4,000 rpm at 4◦C. The aqueous phase was collected and RNA was purified using Direct-Zol RNA miniprep kit (Zymo) according to the manufacturer's instructions. Samples were then treated with DNase Turbo (Ambion) for 1 h and purified with an RNA Clean & Concentrator-25 kit (Zymo) according to the manufacturer's instructions RNA samples from each biological replicate were split in three, in order to generate two 50 -end differentially treated libraries and one RNAseq expression library. RNA for library 1 (“converted” library) was treated either with RNA 50 pyrophosphohydrolase RPPH (NEB) (exponential phase experiment), or with 50 polyphosphatase (Epicenter) (hypoxia experiment and normoxia controls), in order to remove the native 50 triphosphates of primary transcripts, whereas RNA for Library 2 (“nonconverted” library) was subject to mock treatment. Thus, the converted libraries capture both 50 triphosphates (converted to monophosphates) and native 50 monophosphate transcripts, while non-converted libraries capture only native 50 monophosphates. Library construction was performed as described by Shell et al. (2015). For RNA expression libraries, KAPA stranded RNA-Seq library preparation kit and NEBNext Ultra RNA library prep kit for Illumina (NEB) were used for Dataset 1 and Dataset 2, respectively, according to manufacturer's instructions. The following major  odifications were introduced into the protocols: (i) For RNA fragmentation, in order to obtain fragments around 300 nt, RNA was mixed with the corresponding buffer and placed at 85◦C for 6 min (Dataset 1), or at 94◦C for 12 min (Dataset 2). (ii) For library amplification, 10 or 19–23 PCR cycles were used for Dataset 1 and Dataset 2, respectively. The number of cycles was chosen according to the amount of cDNA obtained for each sample.
GEO Accession
GSM3673863