title: GSM3082793: THAP_insoluble_TEDseq_rep5; Homo sapiens; RNA-Seq
library_construction_protocol: Cell fractionation was performed by differential centrifugation following by THAP treatment (2 μM for 2 hr). Briefly, the cells were washed with cold PBS twice and lysed by cold Buffer L (50 mM Tris-HCL (pH7.6), 50 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1 mM β-mercaptoethanol, 1x Roche complete protease inhibitor EDTA-free, 10 μl SUPERaseIN/ml) by scraping and pipetting. Cell lysates were homogenized and centrifuged at 2,000g at 4°C for 2 min. Pellet is nucleus fraction and aqua phase includes cytoplasmic fraction. Cytoplasmic fraction was centrifuged at 10,000g at 4°C for 10 min and insoluble fraction was obtained from the pellet. The insoluble fraction includes stress granules in TAHP-treated cells.  For TED-seq, total RNA was isolated using TRIzol or TRIzol-LS according to the manufacturer's protocol (Invitrogen Life Technologies). Poly(A) RNA was isolated from 5~10 μg of total RNA using Dynabeads® mRNA purification kit (Ambion) according to the manufacturer's protocol.   The ribosome footprint strategy was followed as the previous paper (Ingolia et al., 2012) and partially modified for the generation of RNA-seq library. In brief, cultured cells were washed twice with ice-cold 1x PBS and lyzed by adding lysis buffer (20 mM Tris-Cl (pH7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml cycloheximide, 1% (v/v) Triton X-100, 25 U/ml Turbo DNase I) and triturating the cells ten times through a 26-gauge needle. After centrifuging the cell lysate at 20,000g at 4°C for 10 min, the supernatant was divided into two groups for ribosome profiling library and mRNA expression profiling library.  For ribosome profiling library, RNase I (100 U/μl) was treated in lysate and incubated at room temperature for 45 min. After nuclease digestion, ribosome footprinted RNAs were purified by sedimenting through a 1 M sucrose cushion and excising the urea gel between 26-34 nucleotides.  For mRNA expression profiling library, TRIzol was added into the supernatant and total RNA was extracted according to the manufacturer's protocol.  For nuclei isolation of PRO-seq, cells were washed twice with 1X cold PBS. Cells were scrapped with 1ml of Buffer D (50mM Tris-Cl, pH 8.0, 25% glycerol, 5mM M MgCl2, 0.1mM EDTA, 5mM DTT). Cells were pelleted at 1000 x g for 5min and then resuspended in 50 µl of Buffer D. And then flash frozen in liquid nitrogen. In TED-seq libralies, for 3′ RNA adaptor ligation, 40 pmol of RA3 adaptor (5′phosphate-UGGAAUUCUGGGGUGCCAAGG-/3InvdT/-3′) was treated into each poly(A) RNA and incubated in 20°C for 6 hr. After RNA extraction with TRIzol, RNA fragmentation with 1N NaOH was performed by incubating on ice for 10 min. To remove un-ligated adaptors, poly(A) RNA was isolated from the fragmented RNA using Dynabeads® mRNA purification kit (Ambion). 5′ end phosphorylation was performed using T4 polynucleotide kinase (PNK, NEB) according to the manufacturer's protocol, following by RNA extraction with TRIzol, For 5′ RNA adaptor ligation, 30 pmol of RA5 (5′-GUUCAGAGUUCUACAGUCCGACGAUCNNNNNNNN-3′) and 50 pmol of RT primer (RTP, 5′-GCCTTGGCACCCGAGAATTCCA-3′) were treated into each RNA and incubated in 20°C for 6 hr. 5′-ligated RNA was reverse-transcribed using Superscript II Reverse Transcriptase kit (Invitrogen) including 0.1 M DTT, 12.5 mM dNTP mixture, SUPERaseIN (Ambion). For first size selection of the library, first round amplification was performed with 8 cycles. Following by loading the PCR products to big native 6% polyacrylamide gel, gel bands between 350-360 bp (insert 300 bp) from each sample were excised and then DNA was recovered using Ampure XP beads (Beckman Coulter, Inc.) as provided by the manufacturer's protocol. To obtain precisely size-selected library, recovered DNA was used for second amplification with optimal cycles and different RPI-X barcode index primer according to TruSeq Library Prep Pooling guide (Illumina). Finally, gel extraction was repeated with bands with 426 bp for each selected insert size. The libraries were pooled and sequenced on Illumina NextSeq500 (75 bp) with PhiX control library and the spike-in mixture.
attributes.tag: GEO Accession
attributes.value: GSM3082793