title: GSM1234748: 3T9.mycER.RNAseq.0hOHT_4; Mus musculus; RNA-Seq
library_construction_protocol: B cells: Total RNA was purified using Trizol (Invitrogen), treated with Turbo DNase (Ambion) and purified with RNA Clean XP (Agencourt). 5 mg of purified RNA were then treated with Ribozero rRNA removal kit (Epicentre) and EtOH precipitated. RNA quality and removal of rRNA were checked with the Agilent 2100 Bioanalyser (Agilent Technologies); fibroblasts: RNA was extracted with the Qiagen miRNeasy kit according to the manufacturer. DNA was digested on the columns (Qiagen). 5 μg of purified RNA were then treated with Ribozero rRNA removal kit (Epicentre) and EtOH precipitated. RNA quality and removal of rRNA were checked with the Agilent 2100 Bioanalyser (Agilent Technologies).; samples 28 and 29 (4sU): 4sU-labeling and sequencing of 4sU-labeled RNA of 3T9 MycER fibroblasts was performed as described previously with minor modifications. Briefly, MycER fibroblasts were treated with OHT for 4 hours. Cells were labeled with 300µM of 4-thiouridine (4sU; Sigma T4509) for 10 min. Medium was removed, cells were put on ice, washed 3x with cold PBS. Cells were scraped in cold PBS and pelleted. RNA was extracted with the Qiagen miRNeasy kit according to the manufacturer including the recommended DNase I digest. 50µg of total RNA was used for the biotinylation reaction: RNA was diluted in 100µl of RNAse free water. 100µl of biotinylation buffer (2.5x stock; 25 mM Tris, pH 7.4, 2.5 mM EDTA) and 50µl of EZ-link Biotin-HPDP (1mg/ml in DMF; Pierce/Thermo Scientific 21341) were added and incubated for 2 hours at room temperature. RNA was precipitated and unbound Biotin-HPDP was removed by a combination of chloroform/isamylalcohol (24:1) precipitation with purification using MaXtract high density tubes from Qiagen. Biotinylated RNA was purified using Dynabeads MyOne Streptavidin T1 (Invitrogen). Before addition of RNA, 50µl of beads were washed 2x in washing buffer A (100 mM NaOH, 50 mM NaCl) and once in washing buffer B (100 mM NaCl). Beads were resuspended in 100µl of buffer C (2M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 0.1% Tween-20) to a final concentration of 5 μg/μl. RNA was added in an equal volume and rotated at room temperature for 15 min. Beads were washed 3x with washing buffer D (1M NaCl, 5 mM Tris, pH 7.5, 0.5 mM EDTA, 0.05% Tween-20). RNA was eluted from the beads in 100µl of 10mM EDTA in 95% formamide (65°C, 10 min). RNA was extracted with the RNeasy MinElute Spin columns from Qiagen according to the manufacturer and eluted in 14µl of RNase free water. RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). The 4sU-sequencing library was prepared with the Tru-Seq Illumina kit following the manufacturer's instructions starting from RNA fragmentation. Libraries for RNA-Seq were then prepared with the TruSeq RNA Sample Preparation Kits v2 (Illumina) following manufacturer instruction (except for skipping the first step of mRNA purification with poly-T oligo-attached magnetic beads).
attributes.tag: GEO Accession
attributes.value: GSM1234748