title: Illumina MiSeq sequencing; Determination of the transcription start sites of heterologous promoters in Actinoplanes sp. SE50/110 by 5'-end specific transcriptome sequencing
design: Determination of the transcription start sites of heterologous promoters in Actinoplanes sp. SE50/110 by 5'-end specific transcriptome sequencing
library_construction_protocol: For transcriptome analysis, 2x 1 mL samples from Actinoplanes culture were taken during exponential growth phase, separated from the supernatant by centrifugation (10 s) and snap-frozen in liquid nitrogen. Pellets were stored at -80 °C until further processing. Cultivation was performed in 250 mL Corning® Erlenmeyer baffled cell culture flasks at 28 °C and 140 rpm for seven days in maltose minimal medium (72.06 g?L-1 maltose·1H2O, 5 g?L-1 (NH4)2SO4, 0.184 g?L-1 FeCl2·4H2O, 5.7 g?L-1 Na3C6H5O7·2H2O, 1 g?L-1 MgCl2·6H2O, 2 g?L-1 CaCl2·2H2O, trace elements (final concentration: 1 µM CuCl2, 50 µM ZnCl2, 7.5 µM MnCl2 dissolved in 1 M HCl) and phosphate buffer consisting of 5 g?L-1 each K2HPO4 and KH2PO4 in aqua distilled). The medium was prepared and filter sterilized following the protocol of Wendler et al. (2013). For inoculation of 50 mL medium, 1 mL spore solution of an OD = 3-5 was used. Cell dry weights were determined like described by Wolf et al. (2017). The supernatant was stored for later analysis at -20 °C. For RNA isolation, frozen cell pellets were resuspended in 500 µL LB-buffer (NucleoSpin® RNA Plus, Macherey-Nagel, Düren, Germany) and transferred to 2 mL lysing matrix tubes (0.1 mm spherical silica beads, MP Biomedicals, Santa Ana, California, USA). Cell disruption was carried out in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) for three times 20 s at speed setting 6.5 and 5 min on ice in between. Subsequently, the cell suspension was centrifuged for 5 min at 13,000 g and 4 °C. The supernatant was used for RNA extraction using the NucleoSpin® RNA Plus Kit in combination with rDNase Set (Macherey-Nagel, Düren, Germany) for an on-column DNA digestion. After clean-up and elution according to manufacturer's protocol, the DNA-digestion was repeated in-solution and the sample cleaned up again by use of the same kit. Residual DNA was tested negatively with two primer pairs binding to genomic DNA of Actinoplanes sp. SE50/110 and amplifying small fragments at round about 200-300 nt. The quantity of RNA was analyzed with the NanoDrop 1000 spectrometer (Peqlab, Erlangen, Germany). RNA of triplicates of each mutant were isolated from exponentially grown cultures and equimolar pooled (total amount of 10 µg RNA in 26 µL RNAse-free water). Stable RNA was depleted by use of the Ribo-Zero rRNA Removal Kit for bacteria (Illumina, San Diego, USA). The sample was analyzed by an Agilent RNA 6000 Pico chip in the Bioanalyzer (Agilent, Böblingen, Germany).For fragmentation, 80 µL sample was treated with 20 µL fragmentation buffer (100 mM KOAc, 30 mM MgOAc in 200 mM Tris-HCl pH 8.1) and incubated for 3.5 min at 94 °C. For termination, 100 µL ice-cold fragmentation stop buffer (10 mM Tris, 1 mM EDTA, pH 8) was added and incubated on ice for 5 min. The fragmented RNA was cleaned up and concentrated by use of the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Deutschland), eluted in 18.5 µL RNAse-free water and analyzed in the Bioanalyzer (see above).5?-library preparation was carried out according to the protocol of Pfeifer-Sancar et al. (2013). First, RNA was processed by a terminator exonuclease XRN-1 (NEB, Ipswich, MA, USA) to digest mono- and diphosphorylated transcripts leaving native transcripts with 5? triphosphate end (5?-PPP). Reaction was carried out for 60 min at 37 °C and heat-inactivated for 10 min at 70 °C, according to the manufacturer?s instructions. The library was cleaned up and concentrated by use of RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Deutschland). Second, the 5?-PPP-transcripts were converted to monophosphorylated transcripts (5?-P) by a RNA 5?-Polyphosphatase (Epicentre, Madison, USA). The reaction was performed according to manufacturer?s protocol for 45 min at 37 °C. The processed RNA-pool was precipitated. For this, the volume was adjusted to 180 µL using RNase-free water. 20 µL of 3 M sodium acetate (NaOAc, pH 5.2) and 2 µL glycogen (10 mg mL-1) were added and mixed by gentle vortexing. Precipitation was accomplished by addition of 600 µL ice-cold 100 % ethanol. The sample was inverted several times and stored overnight at -20 °C. The precipitate was centrifuged (>16,000 g at 4 °C for 30 min) and washed twice with 500 µL ice-cold 70 % (v/v) ethanol. The pellet was air-dried and taken up in 13 µL RNAse-free water. Next, adapters were ligated to the 5?-ends of the prepared RNA fragments by addition of 60 µM of RNA adapter (Ligation-5?RNA-Adapter 1: CCCUACACGACGCUCUUCCGAUCGAG) and T4 RNA ligase 1 (NEB, Ipswich, MA, USA) according to manufacturer?s protocol. The library was cleaned up and concentrated by use of the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Deutschland). For reverse transcription and tagging of the 3?-end of the cDNA, a stem-loop DNA adapter was added into the reverse transcription reaction (Loop primer (DNA): AGATCGGAAGAGAGACGTGTGCTCTTCCGATCTNNNNNNN). In advance, the loop primer was denatured for 3 min at 98 °C and cooled to 25 °C at a rate of 1 °C per 10 sec in a Mastercycler pro S (Eppendorf, Hamburg, Germany), like described by Pfeifer-Sancar et al. (2013). For reverse transcription, SuperScriptTM III First-Strand Reverse Transcriptase (Invitrogen, Carlsbad, USA) was used according to manufacturer?s instructions under addition of 5 µM of the prepared loop primer and 1 µL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen, Carlsbad, USA). Reaction was carried out for 30 min at 16 °C, 60 min at 50 °C and inactivated at 85 °C for 5 min. After cDNA synthesis, RNA was digested by RNase H (NEB, Ipswich, MA, USA) for 20 min at 37 °C. The cDNA library was amplified directly by use of the Phusion® High-Fidelity PCR Master Mix with GC Buffer (NEB, Ipswich, MA, USA) (PCR Primer 1 (DNA): AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCGAG, PCR Primer 2.1 (DNA): CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT). The library was cleaned up from a 1.5 % agarose gel (Certified Low Range Ultra Agarose, Bio-Rad, Hercules, USA) cut between 150 to 1,000 bp and purified by the QIAquick Gel Extraction Kit (Qiagen, Hilden, Deutschland). The library was eluted in 20 µL water and quantified by a DNA High Sensitivity Assay chip in the Bioanalyzer (Agilent, Böblingen, Germany).