|
| Status |
Public on May 12, 2021 |
| Title |
Repeat-tagged sfgfp Nanopore Sequencing |
| Sample type |
SRA |
| |
|
| Source name |
Repeat-tagged sfgfp
|
| Organism |
Escherichia coli |
| Characteristics |
promoter: pBAD
library source: transcriptome
|
| Treatment protocol |
RNA samples were treated with DNase I to remove potential DNA contamination. Fragmentation was achieved by addition of a ZnCl2 fragmentation buffer (final concentration: 10 mM Tris-HCl pH 6.8, 10 mM ZnCl2) and heating for 10 min at 95°C. Fragmentation was immediately stopped by putting the sample on ice and addition of 2µl 500 mM Na2-EDTA pH 8. The fragmented RNA was separated by denaturating Urea-PAGE and extracted from the gel. Fragmentation by bivalent metal ions presumably results in 2ʹ,3ʹ-cyclic phosphate and 5ʹ-OH RNA termini (Forconi & Herschlag, 2009), so fragments were first dephosphorylated (for 2ʹ,3ʹ-cyclic phosphate curation) and subsequently phosphorylated (for 5ʹ-OH curation) with T4 PNK. 15 µl of fragmented RNA was mixed with 6 µl of 5x dephosphorylation buffer (500 mM Tris-HCl pH 6.5, 500 MgAc, 25 mM β-mercaptoethanol) and 1 µl T4 PNK (Ambion) in a total volume of 30 µl and incubation for 6 h at 37 °C for dephosphorylation. For phosphorylation, 1 mM ATP and 1 µl T4 PNK were added and the mixture was further incubated for 1 h at 37 °C.
|
| Growth protocol |
Cells were grown in LB medium to logarithmic phase
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The treated RNA was afterwards purified by EtOH precipitation including 1 % (v/v) glycogen.
cDNA libraries were created with the NEBNext® Multiplex Small RNA Library Pret Set for Illumunia according to the manufacturer’s instructions. 100 ng of input RNA was used and amplified cDNA libraries were separated by native PAGE for size selection of 120-250 bp. The selected sizes were extracted from the gel and EtOH precipitated Illumina HiSeq 2500 sequencing were performed at the Max-Planck Genome Centre, Cologne, according to their protocol.
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
MinION |
| |
|
| Description |
Extracted RNA was treated with E. coli Poly(A) Polymerase (NEB) for 30 min 37°C to add a poly(A) tail and then processed for Nanopore sequencing with the Direct RNA Sequencing Kit according to the protocol provided by Oxford Nanopore Technologies.
small RNA
|
| Data processing |
removal of sequences of low quality (quality score limit, 0.05; maximum number of ambiguities 2)
trimming of adapter sequences
filtering by length (15-nt cutoff)
The trimmed sequences were mapped to the reference genome of Bl21 (DE3) and the sequence of the target plasmid using default settings.
Genome_build: E.coli reference genome (CP001509) pETDuet1_sfGFP_Repeat pBAD_sfGFP_Repeat pBAD_lacZ_Repeat
Supplementary_files_format_and_content: The processed data files are in .wig (wiggle) format. They contain the mapping graphs of the three samples which were created from the coverage plots of the generated read track files.
|
| |
|
| Submission date |
May 11, 2021 |
| Last update date |
May 12, 2021 |
| Contact name |
Lennart Randau |
| E-mail(s) |
lennart.randau@staff.uni-marburg.de
|
| Organization name |
Phillips-Universität Marburg
|
| Department |
Biology
|
| Lab |
Randau
|
| Street address |
Karl-von-Frisch-Str. 4
|
| City |
Marburg |
| State/province |
Hessen |
| ZIP/Postal code |
35043 |
| Country |
Germany |
| |
|
| Platform ID |
GPL30119 |
| Series (1) |
|
| Relations |
| BioSample |
SAMN19108933 |
| SRA |
SRX10844226 |
