characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-06-24T17:01:44Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2014-05-07T15:26:11Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2612438
characteristics.characteristic: External Id
characteristics.text: MRC Toxicology Unit, Leicester
characteristics.characteristic: INSDC center name
characteristics.text: 2014-06-24T17:01:44Z
characteristics.characteristic: INSDC first public
characteristics.text: 2014-05-07T15:26:11Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS451363
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2566:rnl2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: sequencing library created using the CircLig protocol with trRnl2 K277Q Protocols: The RNA fragment pool 5' - GCAGUUGCCANNNNNNNNNN - 3' was synthesised (ThermoScientific Molecular Biology). Fragments contained both 5'phosphate and 3'hydroxyl moieties necessary for ligation. 40 ng RNA pool (~6 pmol) and 100 ng Universal Cloning Linker (~18 pmol; NEB) were denatured at 80oC for 2 min then placed immediately on ice. Ligation with 200 U T4 RNA Ligase 2 truncated K227Q mutant (trRnl2 K227Q; NEB) was performed in the presence of 1X RNA ligase buffer (NEB), 15% PEG8000, 20 U SuperaseIn (Ambion), at 25oC for 3 hr. Reactions were terminated by heat inactivation at 90oC and RNA/DNA recovered by isopropanol precipitation with 1 µl GlycoBlue (Ambion) as a coprecipitant. Reaction mixtures were denatured at 80oC in an equal volume of formamide loading buffer (96% Formamide, 10 mM EDTA, 0.01 % bromophenol blue, 0.01 % xylene cyanol) and separated on a 15% TBE-Urea polyacrylamide gel (Invitrogen). 250 ng small RNA marker (Abnova), denatured the same way, was run in adjacent wells. Gels were stained with SybrGold (Invitrogen) and visualised using a Safe Imager (Invitrogen) to guide excision of the 37 nt band. RNA/DNA was recovered from the gel slice by overnight elution in 300 mM NaCl, 0.1% SDS at 4oC followed by isopropanol precipitation and resuspension in 10 μl nucleasefree water. 2 µl of 1.25 µM Ion Torrent compatible reverse transcription (RT) primer (5'CGCCTTGGCC/Sp/CACTCA/Sp/CCTCTCTATGGGCAGTCGGTGATATCTATTGATGGTGCCTACAG - 3'where Sp is an 18-atom hexa-ethyleneglycol spacer) was added to each sample, denatured at 80oC for 3 min then snap cooled on ice. Reverse transcription was performed using 200U Superscript III (Invitrogen) at 48oC for 30 min in 1X first strand buffer (Invitrogen), 0.5 mM dNTPs, 5 mM DTT and 20 U SuperaseIn. RNA was hydrolysed by incubation at 98oC for 20 min with 100 mM NaOH, 50 mM EDTA. cDNA was recovered by isopropanol precipitation and separated on a 6% TBE-Urea polyacrylamide gel. The upper band (~80bp) was excised, being careful to avoid the lower weight bands (unincorporated RT primer and RT primer dimers). cDNA was recovered from the gel slice and circularised with 100 U Circligase (Epicentre) in 1X Circligase buffer, 2.5 mM MnCl2, 0.5 mM ATP, at 60oC for 1 hr. Reactions were heat inactivated at 80oC and cDNA precipitated.
characteristics.characteristic: description
characteristics.text: synthetic construct
characteristics.characteristic: organism
characteristics.text: synthetic construct
characteristics.characteristic: organism
characteristics.text: synthetic construct
characteristics.characteristic: scientific_name
characteristics.text: rnl2
characteristics.characteristic: title