title: NextSeq 500 sequencing; Transcription Start Site (TSS) mapping and total RNAseq of Streptomyces scabies wild-type (WT) versus Streptomyces scabies ΔbtmL
design: Transcription Start Site (TSS) mapping and total RNAseq of Streptomyces scabies wild-type (WT) versus Streptomyces scabies ΔbtmL
library_construction_protocol: 2 mL samples of S. scabies liquid cultures were harvested after 72 h incubation, treated with 1 volume of RNAlater (ThermoFisher Scientific) and stored at -80 °C until extraction. 30 μL of concentrated spores of S. scabies were used to inoculate 10 mL of GYM (ISP2) medium in 50 mL flasks and were incubated for 48 h at 30 °C and 250 rpm. 250 μL of seed culture were used to inoculate 10 mL of bottromycin production medium (BPM) in 50 mL Falcon tubes covered with foam bungs instead of caps. Samples of these cultures were harvested after 72 h cultivation at 30 °C and 250 rpm. The samples for transcription start site (TSS) determination were then with RNA 5' polyphosphatase (5'PP; Epicentre) to convert the 5'-triphosphate groups of primary transcripts into 5'-monophosphate ends amenable for ligation with the alternative tag sequence TAATGCGC. The samples were resuspended in 1 mL of RLT buffer from the RNeasy Kit (Qiagen) and  homogenised in lysing matrix B tubes (MP Biomedicals) using a FastPrep instrument and a program of 3x30 s pulses at 6 m/s with 1 min cooling intervals on ice. The lysates were then centrifuged at 13,000 rpm and 700 μL of supernatant from each sample were transferred to spin tubes from the RNeasy Kit to undergo purification following manufacturer instructions. Chromosomal DNA contamination was eliminated with on-column DNase I treatment (Qiagen) and a further cleanup step using TURBO DNA-free Kit (Ambion, Invitrogen).  The samples were submitted to Vertis Biotechnologie AG (Germany) for the construction and sequencing of tagRNA-seq libraries in a protocol adapted from the technique described in Innocenty et al. (Innocenti et al., 2015). Prior to library construction, rRNA was depleted in the samples using the Ribo-Zero rRNA Removal Kit for bacteria (Epicentre). The remaining material was labelled sequentially as follows: sequence tag CTGAAGCT was ligated to transcripts presenting 5'-monophosphate groups (processed transcripts). The samples were then treated with RNA 5' polyphosphatase (5'PP; Epicentre) to convert the 5'-triphosphate groups of primary transcripts into 5'-monophosphate ends amenable for ligation with the alternative tag sequence TAATGCGC. Once labelled, the samples were used as template for first-strand cDNA synthesis using random hexameric primers. After fragmentation and RNA clean up, Illumina TruSeq sequencing adapters were ligated in a strand specific manner to the 5' and 3' ends of the cDNA fragments. The cDNA was then amplified with a proof reading enzyme to enrich the samples. At this point, it is possible to specifically PCR amplify the 5'-ends which carried the two tag sequences, but that would mean losing the information relating to the rest of the transcriptome. Therefore, a full transcriptome enrichment (for fragments that had Illumina adapters on both ends) was carried out in order to preserve the full coverage of the transcriptome in our libraries, which then allowed us to perform more detailed analyses of gene expression levels in S. scabies genome. The resulting material was purified with the Agencourt AMPure XP Kit (Beckman Coulter Genomics) and analysed by capillary electrophoresis. The cDNA preparations were pooled in equimolar amounts and size selected (240-450 bp range).
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype