GSM2474236: 67NR rep2 (3'-seq); Mus musculus; RNA-Seq
Total RNA was extracted using TRIzol reagent (Invitrogen, Life Technologies). After DNase I treatment (TURBO DNA-free, Ambion) the RNA was quantified using a NanoDrop 2000 instrument (Thermo Scientific). 5 µg of total RNA was used for poly(A)+ RNA purification with MicroPoly(A)Purist™ Kit (Ambion). Poly(A)+ RNA was fragmented at 70°C for 10 min in  RNA Fragmentation buffer (Ambion). After ethanol precipitation, RNA was reverse transcribed using anchored oligo-dT and SMART Scribe RT enzyme (Clontech) allowing to add adapter sequences to the 5’ and 3’ ends of RNA fragments. Libraries were amplified by 13 cycles of PCR with Phusion polymerase (New England Biolabs). A size selection step was done using Agencourt AMPure XP (Beckman Coulter) magnetic beads to obtain fragments of 200–300 bp. Purified libraries were subjected to sequencing using the HiSeq 2000 machine (Illumina). 50 bp reads were obtained by single-end sequencing with P5 Illumina primers.
GEO Accession
GSM2474236