GSM3142511: A6; Mus musculus; RNA-Seq
Total RNA was isolated by chloroform extraction from cell lysates prepared with Trizol (Invitrogen) followed by isopropanol precipitation. mRNA was enriched from total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturers instructions. Eluted mRNA (18µl) was directly subjected to the workflow of the NEBNext Ultra Directional RNA Library Prep Kit for Illumina. For ligation custom adaptors were used  (Adaptor-Oligo 1: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', Adaptor-Oligo 2: 5'-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3'). Indexing was done during the PCR enrichment step using custom amplification primers carrying the index sequence indicated with 'NNNNNN'. (Primer1: Oligo_Seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT, primer2: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT, primer3: CAAGCAGAAGACGGCATACGAGAT NNNNNN GTGACTGGAGTT). After XP beads purification libraries were quantified using Qubit dsDNA HS Assay Kit (Invitrogen). For Illumina flowcell production, samples were equimolarly pooled and distributed on all lanes used for 75bp single read sequencing on Illumina HiSeq 2000.
GEO Accession
GSM3142511