GSM3602326: RNA-seq PhenDC3 rep2; Mus musculus; RNA-Seq
Total genomic DNA was extracted with DNAzol (Invitrogen, ref. 10503027), obtained DNA was fractionated according to the size by sucrose gradient. Nascent DNA was precipitated from fractions containing DNA fragments of 0.5 to 2 kb in size. DNA fragments contaminating NS were eliminated by λ-Exo digestion. DNA fragments were phosphorylated by T4 polynucleotide kinase and digested overnight with 150 U of λ-Exo to eliminate contaminating DNA fragments.  DNA phosphorylation and overnight λ-Exo digestion was repeated twice. A third λ−Exo digestion (100 units) was performed for 2 h at 37°C after the second round of overnight digestion. Controls for each nascent strand purification were prepared by treating a half of NS-containing DNA with 0,1 ug/ul RNase A and 100 mU/ul T2 in pH 5.2 for 2 h at 37°C in order to remove RNA primers prior to the λ-Exo digestion. Total RNA was extracted using the RNeasy Mini Kit (Qiagen; cat 74104) according to the manufacturer protocol DNA Libraries wrere constructed using Illumina TruSeq ChIP Sample Prep Set A (ref 15034288) , RNA libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Preparation Kit, according to the manufacturer's protocols. Libraries were validated using the Fragment Analyzer system with the Standard Sensitivity NGS Fragment Analysis Kit (maker).
GEO Accession
GSM3602326