characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2020-01-21T17:08:23Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2019-10-11T17:06:35Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA6066416
characteristics.characteristic: External Id
characteristics.text: Oxford Nanopore Technologies
characteristics.characteristic: INSDC center name
characteristics.text: 2020-01-21T17:08:23Z
characteristics.characteristic: INSDC first public
characteristics.text: 2019-10-11T17:06:35Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS3867536
characteristics.characteristic: SRA accession
characteristics.text: Dmel_PCS109_1
characteristics.characteristic: Submitter Id
characteristics.text: fruit fly
characteristics.characteristic: common name
characteristics.text: D. melanogaster total RNA isolated from adult W1118 flies according to the following protocol: 1) Add 500 µl of TRIzol to a 1.5 ml Eppendorf with 50 mg of flies and homogenise with a disposable cell pestle. 2) Add 500 µl of TRIzol and invert multiple times to mix. 3) Incubate at room temperature for 15 minutes. 4) Centrifuge at 4oC for 10 minutes at 12000 x g. 5) Transfer supernatant to a new 1.5 ml Eppendorf. 6) Add 200 µl of chloroform and invert to mix. 7) Incubate at room-temperature for 10 minutes. 8) Centrifuge at 4oC during 15 minutes at 10000 x g. 9) Transfer supernatant to a new 1.5 ml Eppendorf tube and add 500 µl of ice-cold isopropanol. 10) Invert several times to mix and incubate 15 minutes at room temperature. 11) Centrifuge at 4oC during 10 minutes at 10000 x g. 12) Discard supernatant and add 1000 ul of 70% ice-cold ethanol. 13) Invert several times to wash the pellet. 14) Centrifuge at 4oC during 5 minutes at 10000 x g. 15) Discard the ethanol and use a sterile wipe to absorb the remaining ethanol from the tube walls. 16) Elute in 200 µl of TE. 150 fM of total RNA was used as a template for reverse transcription for use in the PCS-109 cDNA by PCR sequencing kit (Oxford Nanopore) Following the manufacturer’s instructions (https://community.nanoporetech.com/protocols/cdna-pcr-sequencing_sqk-pcs109/v/PCS_9085_v109_revJ_14Aug2019). Primers were modified so that only the forward primer contained rapid attachment chemistry, resulting in single end adaption of the cDNA representing the 5' end of the RNA molecule (stranded sequencing). For amplification of the first strand cDNA, 12 cycles were used and 100 fM of library was loaded onto a FLO-MIN106 flowcell and sequenced for 48 hrs on the GridION system. Basecalling was performed in real time using guppy 3.4.8. Only a subset of pass reads with mean base quality larger than 7 were uploaded.
characteristics.characteristic: description
characteristics.text: adult
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001272
characteristics.characteristic: dev stage
characteristics.text: Drosophila melanogaster
characteristics.characteristic: organism
characteristics.text: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: W1118
characteristics.characteristic: strain
characteristics.text: D. melanogaster PCS109 cDNA
characteristics.characteristic: title