Illumina HiSeq 2000 paired end sequencing; RNA-seq mouse development
RNA-seq mouse development
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.
Experimental Factor: developmental stage
postnatal day 0.5