GSM3179294: POL-Seq, HuD induced cells, rep 1; Mus musculus; RNA-Seq
CRAC: growing cells were UV crosslinked on ice with λ = 254 nm in Stratalinker 1800 (Stratagene). The cells were lysed and treated with DNase (Promega M610A). Cell lysates were incubated with HA agarose beads (26181 Pierce). Ribonucleoprotein complexes on HA beads were trimmed with 0.5 unit RNaseA+T1 mix (RNace-IT, Stratagene 400720-81) and HuD-RNA complexes were eluted. The eluate was incubated with Ni-NTA Agarose (Ni-NTA Superflow 50% suspension IBA 2-3206-010). RNAs bound to HuD were radiolabelled with 32P-γ-ATP and 3′ miRCat-33 linker ligation was performed. Then RNA ligase 1 and barcoded 5′ linker were added and the reaction mixture HuD-RNA complexes were eluted by incubation with NuPage-Eluition buffer. Protein-RNA complexes were resolved on a 4%–12% Bis-TrisNuPAGE gel (Life Technologies, NP0335) in NuPAGE SDS MOPS running buffer (Life Technologies, NP0001) and transferred to nitrocellulose membrane (GE Healthcare, AmershamHybond ECL). Air-dried membrane was exposed on film o.n. and the radioactive bands corresponding to the HuD complexes were cut out.    POL-Seq and RNA-Seq: cells were treated with cycloheximide and then lysed in 300μl of cold lysis buffer. The lysate was centrifuged at 4 °C for 5min at 20.000 g to pellet cell debris. The cytoplasmic lysates loaded on a linear 15–50% [w/v] sucrose gradient and centrifuged in a SW41Ti rotor (Beckman) for 1 h 40 min at 180.000 g at 4 °C in a Beckman Optima™ Optima XPN-100 Ultracentrifuge. Fractions of 1 ml of volume, were then collected monitoring the absorbance at 254 nm with the UA-6 UV/VIS detector (Teledyne Isco). Sucrose fractions corresponding to polysomes and total RNA were pooled together and the RNA was extracted with phenol/chloroform.    3'end mRNA sequencing: RNAs were isolated using Trizol reagent (Life Technologies) following the manufacturer's instructions. CRAC: RNA was extracted and reverse transcribed. cDNA was amplified and PCR products were precipitated, resuspended and separated on a 2.5% MetaPhoragarose (Lonza). After purification with Gel Extraction Kit with MinElute columns (QIAGEN) the samples were sequenced on Illumina HiSeq 2000 platform.     POL-Seq and RNA-Seq:  libraries were prepared with the Illumina TruSeq mRNA stranded Kit. Samples were sequenced on Illumina HiSeq 2500 platform.    3'end mRNA sequencing: libraries were prepared with the Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit REV. Samples were sequenced on Illumina HiSeq 2500 platform.