Streptomyces formicae
Protocols: Spores of S formicae were inoculated on SFM (soy flour, mannitol) agar and grown at 30 degrees C for 2, 3 or 4 days. Samples were harvested using a sterile spatula from plates with cellophanes and flash frozen in liquid nitrogen for storage at -80 degrees C before extraction. Pellets were thawed and crushed in liquid nitrogen using a sterile pestle and mortar on dry ice. Samples were resuspended in 1 mL RLT Buffer (Qiagen) supplemented with b-mercaptoethanol (10 ul in every 1 ml buffer) and vortexed for 1 minute. Samples were then applied to a QIA-shredder column (Qiagen) and centrifuged for 2 minutes. Flow through was collected (leaving the pellet behind) and mixed with 700 ul acidic phenol-chloroform for 1 minute. Samples were then incubated at room temperature for 3 minutes before centrifuging at 13000 rpm for 20 minutes. The upper phase was collected and mixed with 0.5 volumes of 96% ethanol. This was then applied to an RNeasy Mini spin colunmn (Qiagen) and purified following the manufacturers protocol including on column DNase treatment. Following elution, the Turbo-DNase kit was then used according to the manufacturers protocol and a further Qiagen RNeasey mini clean-up was conducted. Samples were then flash frozen in liquid nitrogen for storage at -80 degrees C. The RNA samples were enriched for Cappable-seq RNA. The following steps were carried out: • Capping of the 5' triphosphorylated RNA with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) (NEB) using the vaccinia capping enzyme (VCE) (NEB) for reversible binding of biotinylated RNA species to streptavidin • Capturing of biotinylated RNA species on streptavidin beads and elution to obtain the 5' fragment of the primary transcripts. In order to be able to identify contamination of the Cappable enriched RNAs with processed transcripts carrying 5'-monophosphate ends, our tagRNA-seq method was used for cDNA synthesis. The Cappable-seq enriched RNAs were first poly(A)-tailed using poly(A) polymerase. Then, the 5' Illumina TruSeq sequencing adapters, which carry sequence tags ATTACTCG and TCCGGAGA (in a proportion of 50% of each adapter), were ligated to the 5' mono-phosphate groups (5'P) of processsed transcripts. The samples were then treated with CapClip Acid Pyrophosphatase (Cellscript) in order to convert 5' triphosphate (5'PPP) structures into 5' monophosphate ends. To the newly formed 5'P groups the 5' Illumina TruSeq sequencing adapters, which carry sequence tag CGCTCATT and GAGATTCC (50% each), were ligated. First-strand cDNA synthesis was performed using an oligo(dT)- adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. For Illumina sequencing, 100 - 300 bp long 5' fragments were isolated from the full-length cDNAs. For this purpose the cDNA preparations were fragmented and the 5'-cDNA fragments were then bound to streptavidin magnetic beads. The bound cDNAs were blunted and the 3' Illumina sequencing adapter was ligated to the 3' ends of the cDNA fragments. The bead bound cDNAs were finally PCR-amplified. For Illumina NextSeq sequencing, the cDNA libraries were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 200 - 500 bp using a preparative agarose gel. An aliquot of the size fractionated cDNA pools was analyzed by capillary electrophoresis (Vertis Biotechnologies)
ENA-FIRST-PUBLIC
2019-05-18T04:03:49Z
ENA-LAST-UPDATE
2019-05-16T15:29:17Z
External Id
SAMEA5612925
INSDC center name
UEA
INSDC first public
2019-05-18T04:03:49Z
INSDC last update
2019-05-16T15:29:17Z
INSDC status
public
Submitter Id
E-MTAB-7975:WT4
broker name
ArrayExpress
genotype
wild type genotype
growth condition
SFM (soy flour, mannitol agar)
sample name
E-MTAB-7975:WT4
scientific_name
Streptomyces formicae