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tag: attribute

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characteristic: ENA-CHECKLIST

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characteristic: ENA-LAST-UPDATE

text: SC

characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

text: NOD/ShiLtJ

characteristic: STRAIN

text: house mouse

characteristic: common name

text: Mus musculus

characteristic: organism

text: Mus musculus

type: has member


text: Homo sapiens

characteristic: ArrayExpress-Species

text: SC

text: public

characteristic: Sample Description

characteristic: Strain

text: Pool1rptsolid-sc-2011-07-06T14:52:31Z-10622

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

characteristic: ENA-FIRST-PUBLIC

characteristic: External Id

text: SC

characteristic: INSDC center name

text: public

text: T2DRVpool_3_PDrpt-sc-2011-07-06T14:52:30Z-6383

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

characteristic: SRA accession

text: T2DRVpool_4a_PDrpt-sc-2011-07-06T14:52:30Z-6384

characteristic: Submitter Id

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool_4b_PDrpt-sc-2011-07-06T14:52:29Z-6385

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool_2_PDrpt-sc-2011-07-06T14:52:28Z-6382

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: Pool3rptsolid-sc-2011-07-06T15:02:26Z-10624

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool4a_PCR-sc-2011-07-06T14:52:25Z-4797

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool4b_PCR-sc-2011-07-06T14:52:25Z-4798

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool2_PCR-sc-2011-07-06T14:52:23Z-4795

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool3_PCR-sc-2011-07-06T14:52:23Z-4796

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool1_PCR-sc-2011-07-06T14:52:22Z-4794

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: Pool4rptsolid-sc-2011-07-06T15:02:27Z-10625

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2Dpool1_sol-sc-2011-07-06T15:02:27Z-30463

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2Dpool3_sol-sc-2011-07-06T15:02:29Z-30465

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2Dpool4_sol-sc-2011-07-06T15:02:29Z-30466

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool1_PCRrpt-sc-2011-07-06T15:02:30Z-31651

text: human

text: Homo sapiens

text: Homo sapiens


text: Homo sapiens

text: SC

text: public

text: T2DRVpool1_PD-sc-2011-07-06T14:52:26Z-4807

text: human

text: Homo sapiens

text: Homo sapiens


text: HeLa

characteristic: CellLine

text: CRUK-CRI

text: public

text: Homo sapiens

characteristic: Organism

text: female

characteristic: Sex

text: ArrayExpress

characteristic: broker name

text: human

text: Protocols: HeLa cells grown in a 10 cm plate were covered with ice-cold PBS buffer and subjected to UV-C irradiation (100 mJ/cm2, Stratalinker 2400). Upon removal of PBS buffer, cells were scraped off and transferred into microtubes (2 ml each). Cells were precipitated by centrifugation for 1 min at 14000 rpm and shock-frozen on dry ice. For magnetic bead preparation, 50 ul of protein A-coated Dynabeads (Invitrogen) were washed 2x with 900 ul lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 1 per cent NP-40, 0.1 per cent SDS, 0.5 per cent Na-Deoxycholate). Dynabeads were resuspended in 200 ul lysis buffer, and 10 ug of hnRNP C antibody (Santa Cruz H-105) were added. After rotation at room temperature for 30-60 min, Dynabeads were washed 2x with lysis buffer and kept in last wash until addition of crosslinked lysate. Pellets were resuspended in 1 ml lysis buffer and sonicated. For partial RNase digestion, RNase I (Ambion) was diluted 1:50 and 1:100 in lysis buffer for high and low RNase treatment, respectively. 10 ul RNase I dilution and 5 ul Turbo DNase (Ambion) were added to the crosslinked lysate and incubated for 3 min at 37C and 800 rpm. Cells were precipitated by two rounds of centrifugation at 4C and 14000 rpm for 10 min followed by careful collection of the supernatant. The supernatant was added to Dynabeads and incubated for 1 h or overnight at 4C and 800 rpm. Dynabeads were washed 2x with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 0,1 per cent SDS, 0.5 per cent Na-Deoxycholate, 1 per cent NP-40) and 1x with PNK wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0,2 per cent Tween-20). For dephosphorylation of 3 prime ends, Dynabeads were resuspended in 2 ul 10x Shrimp alkaline phosphatase buffer (Promega), 17.5 ul H2O and 0.1 ul Shrimp alkaline phosphatase (Promega) and incubated at 37C for 10 min with intermittent shaking (10 sec at 700 rpm followed by 20 sec pause). Samples were washed 2x with high-salt wash buffer, 1x with 900 ul PNK wash buffer and 1 x with 50 ul 1x RNA ligase buffer (NEB, freshly prepared from frozen stock). For RNA linker ligation, Dynabeads were resuspended in 15 ul L3 ligation mix (5 ul L3 RNA linker [5 prime- phosphate-UGAGAUCGGAAGAGCGGTTCAG-3 prime-Puromycin, 20 pM], 1.5 ul 10x RNA ligase buffer, 7.75 ul H2O, 0.5 ul RNasin [Promega], 0.25 ul RNA ligase [NEB]) and incubated overnight at 16C. Samples were mixed with 5 ul NuPAGE loading buffer (Invitrogen), incubated for 5 min at 70C and placed on a magnetic stand to collect the eluate. Samples were run on 9-well or 10-well Novex NuPAGE 4-12per cent Bis-Tris gels (Invitrogen) with 1x MOPS running buffer (Invitrogen). After gel electrophoresis, protein and covalently bound RNAs were transferred to a nitrocellulose membrane (Whatman) using a Novex wet transfer apparatus (Invitrogen). The nitrocellulose membrane was rinsed with 1x PBS, wrapped into cling film and exposed to a BioMax XAR Film (Kodak) at -80C. For isolation of crosslinked RNAs, 2 mg/ml proteinase K (Roche) was pre-incubated in PK buffer (100 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM EDTA) for 5 min at 37C. In order to recover different size fractions of RNAs, two fragments were cut out of the nitrocellulose membrane at different heights above the molecular weight of the protein (40 kDa). 200 ul proteinase K solution was added to each fragment and incubated for 30 min at 55C. Incubation was repeated after addition of 130 ul PK/7 M urea buffer (100 mM Tris-HCl pH 7,5, 50 mM NaCl, 10 mM EDTA, 7 M urea). Samples were cooled to 37C, mixed with 170 ul H2O and 600 ul RNA phenol/CHCl3 (Ambion) and incubated for 5 min at 37C and 1100 rpm. After centrifugation for 10 min at 13000 rpm and room temperature, 450ul of the aqueous phase were transferred into a new microtube and again subjected to centrifugation. 400 ul of supernatant were mixed with 0.5 ul Glycoblue (Ambion), 40 ul 3 M sodium acetate pH 5.5 and 1 ml 100 per cent EtOH and incubated overnight at -20C. RNAs were precipitated by centrifugation for 30 min at 15000 rpm and 4C, washed with 500 ul 80 per cent EtOH and resuspended in 12 ul H2O. For reverse transcription, 1 ul RT primer (2 pmol/ul; the following three primers were used for replicates 1 to 3: 5 prime-phosphate- NNNCAAGATCGGAAGAGCGTCGTGGATCCTGAACCGCTC-3 prime; 5 prime-phosphate- NNNGAAGATCGGAAGAGCGTCGTGGATCCTGAACCGC-3prime; 5 prime-phosphate- NNNTGAGATCGGAAGAGCGTCGTGGATCCTGAACCGCTC-3 prime; NNN represents 3-nt random barcode and bold nucleotides mark 2-nt barcode used as an experiment identifier) and 1 ml 10 mM dNTP mix were added to the RNA, preheated for 5 min to 70C and then held at 42C. Once 6 ul RT mix (5 ul 5x RT buffer [Invitrogen], 1 ul 0.1 M DTT, 0.5 ul Superscript III reverse transcriptase [Invitrogen], 0.5 ul RNasin) were added and mixed by pipetting, reverse transcription was performed with the following program: 10 min at 42C, 40 min at 50C, 20 min at 55C, and hold at 4C. To remove RNA, samples were heated for 2 min to 95C, mixed with 1 ul RNase A (Ambion) and incubated for 20 min at 37C. cDNAs were precipitated by addition of 80 ul TE buffer, 0.5 ul Glycoblue, 10 ul 3 M sodium acetate pH 5.3 and 250 ul 100 per cent EtOH, incubation for 1 h on dry ice or overnight at -20C, and centrifugation for 30 min at 4C and 15000 rpm. Pellets were washed with 500 ul 80 per cent EtOH, dried for 3 min at room temperature and resuspended in 6 ul H2O. For size separation, cDNAs were mixed with 2 ul 2x TBE-urea loading buffer (Invitrogen) and incubated for 3 min at 70C. Samples were run on a 6 per cent TBE urea gel (Invitrogen) in 1x TBE buffer for 40 min at 180 V. In order to recover different size fractions, two bands were cut from the gel corresponding to a cDNA size of 100 - 175 nt and 175 - 350 nt. Gel fragments were mixed with 400 ml TE buffer, crushed with a 1 ml syringe plunger and incubated for 2 h at 37C and 1100 rpm. A Costar SpinX column (Corning Incorporated) was prepared by addition of two 1 cm glass wool pre-filters (Whatman 1823-101) and centrifugation for 1 min at 13000 rpm. After transfer of the supernatant to the column, 40ul 3 M sodium acetate pH 5.5 and 0.5ul glycogen were added. Columns were vortexed before adding 1 ml 100 per cent EtOH and incubating overnight at -20C. Columns were washed by addition of 500ul 80 per cent EtOH and centrifugation for 10 min at 15000 rpm and 4C. Pellets were dried for 3 min at room temperature and resuspended in 12ul H2O. In order to circularize the cDNAs, samples were mixed with 1.5ul 10x CircLigase buffer II (Epicentre), 0.75ul 50 mM MnCl2 and 0.75ul CircLigase II (Epicentre) and incubated for 1h at 60C. For subsequent linearization, a primer (5 prime- GTTCAGGATCCACGACGCTCTTCAAAA-3 prime) complementary to the BamHI restriction site in the RT primer was annealed by adding 26ul H2O, 5ul FastDigest buffer (Fermentas) and 1ul 10uM primer and incubation with the following program: 2 min at 95C, 70 cycles starting for 1 min at 95C and reducing the temperature with every cycle by 1C. BamHI cleavage was performed by adding 3ul Fastdigest BamHI (Fermentas) and incubating for 30 min at 37C. Samples were mixed with 50ul TE buffer, 0.5ul Glycoblue, 10ul 3 M sodium acetate pH 5.5 and 250ul 100 per cent EtOH and incubated for 1 h on dry ice or overnight at -20C. cDNAs were precipitated by centrifugation for 30 min at 15000 rpm and 4C, washed with 500ul 80 per cent EtOH, dried for 3 min at room temperature and resuspended in 9ul H2O.

characteristic: description

text: Homo sapiens

text: Homo sapiens


text: SC

characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

text: human

text: Homo sapiens

text: Homo sapiens


E-MTAB-308:Zebrafish embryo 5 dpf 2

text: 5 days

characteristic: Age

text: embryo

characteristic: DevelopmentalStage

characteristic: ENA-LAST-UPDATE

text: SC

text: public

text: fertilization

characteristic: InitialTimePoint

text: Danio rerio

characteristic: OrganismPart

text: Tuebingen

characteristic: StrainOrLine

text: E-MTAB-308:Zebrafish embryo 5 dpf 2

text: ArrayExpress

text: zebrafish

characteristic: common name

text: Protocols: Zebrafish embyos or tissues were collected from a Tuebingen strain incross and grown at 28 C. Collected samples were snap frozen on dry ice and stored at -70 C Total RNA was extracted using Trizol Reagent (Invitrogen) following the manufacturer's instructions. Pellets were resuspended RNase-free 10 mM Tris pH 7.5 and the RNA was quantified using a NanoDrop ND-1000 Spectrophotometer (Axon Instruments).

text: Danio rerio

characteristic: organism

text: Danio rerio


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: External Id

text: UNIVERSITY OF ALASKA FAIRBANKS

characteristic: INSDC center name

text: public

characteristic: SRA accession

tag: Namespace:UNIVERSITY OF ALASKA FAIRBANKS

characteristic: Submitter Id

text: marine metagenome

characteristic: depth

text: Arctic sea ice metagenome, 1.1 m

text: marine biome

characteristic: environment (biome)

text: marine water body

characteristic: environment (feature)

text: sea ice

characteristic: environment (material)

characteristic: event date/time

characteristic: event label

text: Arctic Ocean

characteristic: geographic location (country and/or sea)

characteristic: geographic location (latitude)

characteristic: geographic location (longitude)

characteristic: last update date

text: Arctic Ocean

characteristic: marine region

text: marine metagenome

text: GEOTRACES ARCTIC 2015

characteristic: project name

characteristic: protocol label

characteristic: salinity

text: ice auger

characteristic: sample collection device

text: GEOTRACES ARCTIC 2015

characteristic: sampling campaign

text: CGC Healy

characteristic: sampling platform

characteristic: sampling station

text: marine metagenome

text: Illumina MiSeq 2x250 PE

characteristic: sequencing method

text: 0.22 um

characteristic: size fraction lower threshold

characteristic: size fraction upper threshold

characteristic: temperature

text: Arctic sea ice metagenome, 1.1 m

characteristic: title


characteristic: External Id

text: SC

characteristic: INSDC center name

characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

characteristic: Submitter Id

text: barber pole worm

characteristic: common name

text: Haemonchus contortus

characteristic: organism

text: Haemonchus contortus