characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-08-08T17:01:13Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:11:12Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2361854
characteristics.characteristic: External Id
characteristics.text: Cambridge Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2014-08-08T17:01:13Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:11:12Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS406880
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2328:P4_l_02
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: C57BL/6J mice were bred and housed under UK Home Office licensing husbandry standards. After breeding, mice were observed for plugs. Morning of day when plug was first observed was counted as day E0. Samples were prepared by direct perfusion of the liver with buffered salt solution. Brain and liver tissues were minzed and fresh-frozen in liquid nitrogen. Total RNA was extracted from liver and brain tissues of all developmental stages. Excise 25 mg liver and brain tissue sample (on dry ice) into Precellys tubes CK28-R, bertin technology. Remove the sample tube from the dry ice and follow the instructions of the RNeasy Min Kit (Qiagen). Add Lysis Reagent into each sample tube. Homogenize immediately on the TissueLyser for 2x10 5000 with 10 break. After pure RNA is obtained (RNeasy Min Kit (Qiagen) instruction), Turbo DNAse treat (Ambion AM1907) 10ug total RNA, 1/10th volume DNase buffer (10ul), Turbo DNase enzyme (2ul), Water (to 100ul) Incubate 37C 30 minutes (mix inactivation agent) 0.15 volume inactivation agent (15ul). Ribosomal RNA of 5 ug of DNase-treated total RNA was removed using the RiboZero (Epicentre RZC110424) kit following manufacturer's instructions. First Strand cDNA synthesis was performed using the Invitrogen it (11917-010) following manufacturer's instructions. Second strand cDNA synthesis was done by using a 10mM dUTP mix (Promega U1335) (dATP, dCTP, dGTP, dUTP). Make up reaction on ice Volume cDNA from step 3 50ul 5x FSS buffer, 4ul DTT 2ul, 5xSSS buffer 30ul DU/NTP mix 3ul, E.coli DNA ligase 1ul, E.Coli DNA polymerase 3ul, E.Coli Rnase H 1ul, Water 56ul, Incubate 16C for 2 hours, Add 2ul T4 DNA polymerase 5 minutes 16C. Qiagen column purification column Elute 50ul water or EB. Library was prepared following Illumina TruSeq RNA sample prep Low Throughput kit instructions with the exception to treat sample with 1ul Uracil N-glycosylase (UNG) 37C 15minutes (Applied Biosystems) before PCR amplification.
characteristics.characteristic: description
characteristics.text: postnatal day 4
characteristics.characteristic: developmental stage
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: liver
characteristics.characteristic: organism part
characteristics.text: wild type
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: phenotype
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: C57BL/6J
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000606
characteristics.characteristic: strain
characteristics.text: P4_l_02
characteristics.characteristic: title