title: GSM3525841: CAST.B6_E7.5_ExE_RNA_1; Mus musculus; RNA-Seq
library_construction_protocol: Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5).  Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated.  Single E7.5 epiblast and ExE samples were individually frozen in 10µL of buffer RLT Plus (Qiagen). Total RNA was extracted using a trizol extraction method, as previously described (Veselovska et al. 2015).  In brief, samples were homogenized in 400µL of TRIzol (Invitrogen) and phase separated by adding 80µL of chloroform:isoamyl alcohol (Sigma), mix and centrifuge at 4°C for 15 minutes.  The aqueous phase was transferred to a new tube, 1uL GlycoBlue and 300µL of ice cold isopropanol was added and mixed.  Samples were incubated for 10 minutes and then centrifuged for 10 minutes at 4°C.  The pellet was washed once with 75% ethanol, air dried and then resuspended in 5µL of RNase-free water.  20µL of lysis/binding buffer was immediately added to each RNA sample and oligo(dT)25 capture of mRNA was done using Dynabeads mRNA DIRECT kit (Life Techologies).  The protocol was implemented as per manufacturers' instructions including the additional steps for elimination of rRNA contamination. Maxymum Recovery tubes (Axygen) were used and volumes were adapted for the low amount of starting material: 5µL of Dynabeads Oligo(dT)25 were used for each sample, mRNA capture was done in a total volume of 50µL of lysis/binding buffer, washes were done using 100µL Washing Buffer A or 50µL of Washing Buffer B, and a final elution volume of 5µL 10mM Tris-HCl.  The total volume of mRNA was then immediately advanced into the library preparation protocol, using the SMARTer Stranded RNA-seq kit (Clontech), which is optimised for as little as 100pg of RNA.  The protocol was completed as per the manufacturers' instructions and 14 amplification cycles were used for all samples. The quantification of all libraries was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA).  Libraries were sequenced using 50bp single-end on the Illumina HiSeq 2500 RapidRun, multiplexing 12 samples per lane.
attributes.tag: GEO Accession
attributes.value: GSM3525841