GSM4492173: 5377_1_CONT_TEX_2_ACAGTG_S25_R1_001.fastq.gz; Homo sapiens; RNA-Seq
Total RNA: RNA was extractedusingTrizol reagent (15596-018, Ambion life technologies) according to the manufactures protocol followed by DNase I treatment.  PolyA selected RNA was isolated using Oligotex kit (QIAGEN). 5′- CAP RNA immunoprecipitation was performed as described previously (Malka, Y., et al., Post-transcriptional 3 -UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments. Nat Commun, 2017. 8(1): p. 2029.). for 5' end sequencing: 20 μg of total RNA was polyA selected and then was treated with E.coli purified  AlkB for demethylation of the RNA as previously described in (Zheng, G., et al., Efficient and quantitative high-throughput tRNA sequencing. Nat Methods, 2015. 12(9): p. 835-837.).  3′-RNA capping and pulldown was performed as described previously (Malka, Y., et al., Post-transcriptional 3 -UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments. Nat Commun, 2017. 8(1): p. 2029.). Polysomal RNA isolatiowas performed as described previously (Slobodin, B., et al., Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation. Cell, 2017. 169(2): p. 326-337 e12.). Briefly, sucrose gradients for separation of polysomes were usually prepared by gentle sequential addition of 2.2ml of the different sucrose solutions (e.i., 47, 37, 27, 17 and 7% in Tris-HCl pH = 7.5 (f.c. 20mM), MgCl2 (f.c. 10mM) and KCl (f.c. 100mM), supplemented with 2mM DTT, Ribosafe RNAse inhibitor (Bioline, 1 μl/ml) and CHX (100 μg/ml) into a 12 mL tube (Beckman, 9/16 × 3 1/2 in.) and left overnight at 4°C to achieve continuous gradient prior to the centrifugation. Cells were treated with CHX and harvest after washing with PBS with CHX and lysed. The lysates were centrifuged 1300xg for 10 min at 4°C and the supernatants were transferred into new tubes. From the cleared lysates, 500 μL were loaded on top of each gradient, mounted on SW41TI rotor and centrifuged at 36000rpm for 2 hr at 4°C. Following the centrifugation, each gradient was split into 15 equal fractions of 760 μl. Fractions 9-13 were collected for RNA isolation using TRIsure (Bioline) according to the manufacturer's instructions. Total RNA: SMARTer® Stranded RNA-Seq Kit (Takara; 634839) or 3′–3′-mRNA-Seq Library Prep Kit (lexogen; 015UG009V0211). 5' seq: uncapped 5′-specific sequencing libraries as previously described in (Pelechano, V., W. Wei, and L.M. Steinmetz, Genome-wide quantification of 5'-phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics. Nat Protoc, 2016. 11(2): p. 359-76.). Polysome: polyA selection was followed by RNA library preparation as previously described (Takara; 634839). Ribosomal sequencing: Libraries from cultured cells were prepared as described previously (Loayza-Puch, F., et al., p53 induces transcriptional and translational programs to suppress cell proliferation and growth. Genome Biol, 2013. 14(4): p. R32.). For the mouse Tcells Total RNA of 5913 was used for poly A selection and normalization of 5915 samples and Total RNA of 5522 samples was used for selection and normalization of 5522 polysome samples. Total RNA-seq, Ribo-seq, 3' end RNA-seq, 5' RNA-seq, Polysome sequencing
GEO
GSM4492173
GEO Accession
GSM4492173