title: GSM1880854: shMenin_4sU_stranded_UT; Mus musculus; OTHER
library_construction_protocol: For sequencing of ribosomal-RNA depleted total RNA, RNA was isolated with RNeasy (Qiagen) with DNase I treatment. Then, the Illumina kit TruSeq stranded total RNA library preparation kit (RS-122-2201) was used for library synthesis. Briefly, after depletion of ribosomal RNA (Ribo-Zero), the RNA molecules are fragmented, followed by reverse transcription into cDNA with random primers in the presence of actinomycin D. Then, second strand cDNA synthesis is performed with DNA Polymerase I with dUTP instead of dTTP. Hence, the second strand will not be copied and amplified in the PCR-amplification step as the polymerase cannot go past this nucleotide. Then a single A-base is added to the 5' ends and adapters are ligated. The cDNA is a amplified with PCR (15 cycles) to generate the final cDNA library. In order to obtain nascent 4-thio-Uridine (4sU) labelled RNA, 4sU was added to the cell culture medium at a final concentration of 300 mM for 45 min before harvesting the cells. The 4sU-labeled RNA was isolated as described (Rabani M. et al., 2011). Briefly, RNA was isolated with Trizol (Invitrogen), then 50 ug of total RNA was labeled with biotin (EZ-link HPDP-biotin, Thermo Scientific), and unbound biotin was removed with MaXtract high density tubes (Qiagen). Biotinylated RNA was isolated with MyOne Streptavidin T1 Dyneabeads (Invitrogen) for 15 min at room temperature, followed by washing of the beads according to manufacturers instructions. The 4sU-labelled RNA was finally eluted with 95% Formamide (Sigma) in 10 mM EDTA at 65¡C for 10 min, and purified on RNeasy MinElute spin columns (Qiagen), and eluted in 14 ul H2O. A total of 40-100 ng of 4sU-labelled RNA was used for cDNA-library synthesis using Illumina kits, skipping the step of either ribosomal depletion or polyA-selection, starting from the RNA fragmentation step. TruSeq RNA Sample Preparation kit (RS-122-2001) or the kit processing the RNA to retain strand information (Truseq Stranded Total RNA Sample Preparation kit (RS-122-2201), or in the Epicentre ScriptSeq RNA-Seq Library Preparation Kit (SSV21124) was used (for second and first strand synthesis respectively). The Illumina stranded RNAseq kit are based on the dUTP method as described in Levin et. al, Nat. Methods, 2010.
attributes.tag: GEO Accession
attributes.value: GSM1880854