GSM5229840: SGs_6_T; Homo sapiens; RNA-Seq
G3BP1-GFP- U20S cells were grown O/N on 15cm dishes 80-90% confluent and incubated with 8ml of virus free supernatant for 1h, then washed twice with warm DMEM and scraped out and spun down at 1,500g 10 min. The cell pellet was either snap freeze or resuspended in 1ml of lysis buffer (50mM Tris HCl pH 7.4, 100mM Potassium Acetate, 2mM Magnesium Acetate, 0.5mM DTT, 0.5% NP40, 1 complete mini EDTA free protease inhibitor tablet/ 50ml of lysis buffer) and syringe lysis using 25G 5/8 needle on ice seven time and spun at 300g, for 5min at 4C. The supernatant was transferred and centrifuged again at 18000g for 20min at 4C. The supernatant was discarded the pellet was resuspend pellet in 1ml of SG lysis buffer and spun again at 18000g for 20min at 4C. The pellet was resuspended in 300ul lysis buffer (granule-enriched fraction). The RNA was extracted with Trizol following the manufactural procedure. The precipitated RNA was used by Novogene to constitute the cDNA library using oligo(dT) beads, then was then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library is ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. The Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/μl before checking insert size on an Agilent 2100 and quantifying by quantitative PCR (Q-PCR) (library activity >2 nM). Libraries were fed into Illumina machines according to activity and expected data volume.