GSM1810506: RNA-seq, mESCs, LIF+BMP-4; Mus musculus; RNA-Seq
RNA was isolated using the RNeasy Kit (Qiagen). Directional RNA-seq libraries were prepared according to a slightly modification of the protocol provided by Illumina. Briefly, 10 μg of total RNA were subjected to two rounds of oligo-dT purification using Dynabeads Oligo(dT)25 (Invitrogen/Life Technologies). The purified RNA was decapped with Tobacco Acid Pyrophosphatase (TAP) (Epicentre), followed by extraction with phenol:chloroform:isoamyl alcohol (PCIA) precipitation. 100 ng of poly(A)+ RNA were fragmented with 10x fragmentation buffer (Ambion/Life Technologies). Decapped and fragmented RNA was treated with Antarctic phosphatase (New England Biolabs) and T4 polynucleotide kinase (New England Biolabs). The modified RNA was cleaned up with an RNeasy MinElute kit (Qiagen). Libraries were constructed using the Illumina Small RNA sample preparation kit with the v1.5 sRNA 3'adaptor (Illumina).