title: GSM4260775: R1 total cell ribo- RNA-seq replicate2; Mus musculus; OTHER
library_construction_protocol: Total RNA from each cultured cell line or cultured cells with different treatments was extracted with Trizol Reagent (Invitrogen) according to the manufacturer's protocol. Fractionation of the cytoplasmic and nuclear RNA in H9 and R1 cells was performed as following. 1 × 106 cells were rinsed twice with ice-cold PBS, and centrifuged at 1,000 rpm for 3 min. Cell pellet was suspended by gentle pipetting in 200 mL lysis buffer (10 mM Tris pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% Igepal, 2 mM Ribonucleoside Vanadyl Complex), and incubated on ice for 5 min. During the incubation, one fifth of the lysate was added to 1 mL Trizol for total RNA extraction. The rest of the lysate was centrifuged at 1,000 g for 3 min at 4°C to pellet the nuclei and the supernatant was the cytoplasmic fraction. To obtain pure cytoplasmic RNA, the supernatant fraction was centrifuged at 14,000rpm for 10 min at 4°C and then collected the supernatant carefully to a new tube and extracted the RNA with Trizol. To obtain pure nuclear RNA, nuclear pellets underwent additional wash with 200 µL lysis buffer, one additional wash by adding 0.5% deoxycholic acid into the lysis buffer. Finally, the purified nuclei were resuspended in 200 µL lysis buffer followed by extraction with Trizol. Fractionated RNA from the same amount of cells was used for cDNA production, RT-PCR and RNA-seq analysis. For RNA-seq samples from H9 and R1 cells, total/cytoplasmic/nuclear RNA was processed with the RiboMinus kit (Human/Mouse Module, Invitrogen) to deplete ribosomal RNAs for sequencing (Ribo– RNA-seq). For hFAST KD total RNA-seq samples, polyadenylated (poly(A)+) RNA preparation was carried out as following. Briefly, total RNAs (8 μg) were incubated with oligo(dT) magnetic beads to isolate poly(A)+ RNAs, which were bound to beads, selection was performed three times to ensure the purity of poly(A)+ RNA populations. Ribo– and poly(A)+ RNA-seq libraries were prepared using Illumina TruSeq Stranded Total RNA LT Sample Prep Kit. All libraries were size-selected with AmpureXP beads (Agencourt) and quantified using Agilent Bioanalyzer 2100 and QuBit high-sensitivity dsDNA assay. Size-selected libraries were subjected to deep sequencing with Illumina HiSeq X ten at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China. Raw read qualities were evaluated by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
attributes.tag: GEO Accession
attributes.value: GSM4260775